Histone deacetylase inhibitors

ABSTRACT

The invention relates to hydroxamic acid derivatives having carbamate linkage with the structural formula 
                         
that are inhibitors of histone deacetylase (HDAC), and are useful in the prevention and/or treatment of cellular proliferative diseases, for example cancer, autoimmune, allergic and inflammatory diseases, diseases of the central nervous systems (CNS) such as neurodegenerative diseases, and in the prevention and/or treatment of restenosis.

PRIORITY CLAIM

This application is a §371 National Stage Application ofPCT/US2005/029913, filed on Aug. 22, 2005, which claims priority fromU.S. Provisional Application Ser. No. 60/604,469, filed on Aug. 25,2004.

BACKGROUND OF THE INVENTION

This invention relates to hydroxamic acid derivatives having a carbamatelinkage, that are inhibitors of histone deacetylase (HDAC), and areuseful in the prevention and/or treatment of cellular proliferativediseases, for example cancer, autoimmune, allergic and inflammatorydiseases, diseases of the central nervous system (CNS) such asneurodegenerative diseases, and in the prevention and/or treatment ofrestenosis.

Compounds having a hydroxamic acid moiety have been shown to possessuseful biological activities. For example, many peptidyl compoundspossessing a hydroxamic acid moiety are known to inhibit matrixmetalloproteinases (MMPs), which are a family of zinc endopeptidases.The MMPs play a key role in both physiological and pathological tissuedegradation. Therefore, peptidyl compounds that have the ability toinhibit the action of MMPs show utility for the treatment or prophylaxisof conditions involving tissue breakdown and inflammation. Further,compounds having a hydroxamic acid moiety have been shown to inhibithistone deacetylases (HDACs), based at least in part on the zinc bindingproperty of the hydroxamic acid group.

The inhibition of HDACs can repress gene expression, includingexpression of genes related to tumor suppression. Inhibition of histonedeacetylase can lead to the histone deacetylase-mediated transcriptionalrepression of tumor suppressor genes. For example, inhibition of histonedeacetylase can provide a method for treating cancer, hematologicaldisorders, such as hematopoiesis, and genetic related metabolicdisorders. More specifically, transcriptional regulation is a majorevent in cell differentiation, proliferation, and apoptosis. There areseveral lines of evidence that histone acetylation and deacetylation aremechanisms by which transcriptional regulation in a cell is achieved(Grunstein, M., Nature, 389: 349-52 (1997)). These effects are thoughtto occur through changes in the structure of chromatin by altering theaffinity of histone proteins for coiled DNA in the nucleosome. There arefive types of histones that have been identified. Histones H2A, H2B, H3and H4 are found in the nucleosome and H1 is a linker located betweennucleosomes. Each nucleosome contains two of each histone type withinits core, except for H1, which is present singly in the outer portion ofthe nucleosome structure. It is believed that when the histone proteinsare hypoacetylated, there is a greater affinity of the histone to theDNA phosphate backbone. This affinity causes DNA to be tightly bound tothe histone and renders the DNA inaccessible to transcriptionalregulatory elements and machinery.

The regulation of acetylated states occurs through the balance ofactivity between two enzyme complexes, histone acetyl transferase (HAT)and histone deacetylase (HDAC).

The hypoacetylated state is thought to inhibit transcription ofassociated DNA. This hypoacetylated state is catalyzed by largemultiprotein complexes that include HDAC enzymes. In particular, HDACshave been shown to catalyze the removal of acetyl groups from thechromatin core histones.

It has been shown in several instances that the disruption of HAT orHDAC activity is implicated in the development of a malignant phenotype.For instance, in acute promyelocytic leukemia, the oncoprotein producedby the fusion of PML and RAR alpha appears to suppress specific genetranscription through the recruitment of HDACs (Lin, R. J. et al.,Nature 391:811-14 (1998)). In this manner, the neoplastic cell is unableto complete differentiation and leads to excess proliferation of theleukemic cell line.

U.S. Pat. Nos. 5,369,108, 5,932,616, 5,700,811, 6,087,367 and 6,511,990,the contents of which are hereby incorporated by reference, disclosehydroxamic acid derivatives useful for selectively inducing terminaldifferentiation, cell growth arrest or apoptosis of neoplastic cells. Inaddition to their biological activity as antitumor agents, thesehydroxamic acid derivatives have recently been identified as useful fortreating or preventing a wide variety of thioredoxin (TRX)-mediateddiseases and conditions, such as inflammatory diseases, allergicdiseases, autoimmune diseases, diseases associated with oxidative stressor diseases characterized by cellular hyperproliferation (U.S.application Ser. No. 10/369,094, filed Feb. 15, 2003, the entire contentof which is hereby incorporated by reference). Further, these hydroxamicacid derivatives have been identified as useful for treating diseases ofthe central nervous system (CNS) such as neurodegenerative diseases andfor treating brain cancer (See, U.S. application Ser. No. 10/273,401,filed Oct. 16, 2002, the entire content of which is hereby incorporatedby reference).

The inhibition of HDAC by the hydroxamic acid containing compoundsuberoylanilide hydroxamic acid (SAHA) disclosed in the above referencedU.S. patents, is thought to occur through direct interaction with thecatalytic site of the enzyme as demonstrated by X-ray crystallographystudies (Finnin, M. S. et al., Nature 401:188-193 (1999)). The result ofHDAC inhibition is not believed to have a generalized effect on thegenome, but rather, only affects a small subset of the genome (Van Lint,C. et al., Gene Expression 5:245-53 (1996)). Evidence provided by DNAmicroarrays using malignant cell lines cultured with a HDAC inhibitorshows that there are a finite (1-2%) number of genes whose products arealtered. For example, cells treated in culture with HDAC inhibitors showa consistent induction of the cyclin-dependent kinase inhibitor p21(Archer, S. Shufen, M. Shei, A., Hodin, R. PNAS 95:6791-96 (1998)). Thisprotein plays an important role in cell cycle arrest. HDAC inhibitorsare thought to increase the rate of transcription of p21 by propagatingthe hyperacetylated state of histones in the region of the p21 gene,thereby making the gene accessible to transcriptional machinery. Geneswhose expression is not affected by HDAC inhibitors do not displaychanges in the acetylation of regional associated histones (Dressel, U.et al., Anticancer Research 20(2A): 1017-22 (2000)).

Further, hydroxamic acid derivatives such as SAHA have the ability toinduce tumor cell growth arrest, differentiation and/or apoptosis(Richon et al., Proc. Natl. Acad. Sci. USA, 93:5705-5708 (1996)). Thesecompounds are targeted towards mechanisms inherent to the ability of aneoplastic cell to become malignant, as they do not appear to havetoxicity in doses effective for inhibition of tumor growth in animals(Cohen, L. A. et al., Anticancer Research 19:4999-5006 (1999)).

In view of the wide variety of applications for compounds containinghydroxamic acid moieties, the development of new hydroxamic acidderivatives having improved properties, for example, increased potencyor increased bioavailability is highly desirable.

SUMMARY OF THE INVENTION

This invention relates to hydroxamic acid derivatives having a carbamatelinkage, that are inhibitors of histone deacetylase (HDAC), and areuseful in the prevention and/or treatment of cellular proliferativediseases, for example cancer, autoimmune, allergic and inflammatorydiseases, diseases of the central nervous system (CNS) such asneurodegenerative diseases, and in the prevention and/or treatment ofrestenosis.

In a first embodiment, the compounds of the invention may be illustratedby Formula I:

DETAILED DESCRIPTION OF THE INVENTION

The compounds of this invention are useful as HDAC inhibitors and areillustrated by a compound of Formula I:

wherein

R¹ and R² are, independently of each other, unsubstituted or substitutedand selected from C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, cycloalkyl, aryl,heterocyclyl, heteroaryl, C₁-C₁₀ alkyl-C₂-C₁₀ alkenyl, C₁-C₁₀alkylcycloalkyl, C₁-C₁₀ alkylaryl, C₁-C₁₀ alkylheterocyclyl and C₁-C₁₀alkylheteroaryl; R³ and R⁴ are independently hydrogen or C₁-C₁₀ alkyl;and

n is 4, 5, 6 or 7;

wherein when R¹ is phenyl or 8-quinolinyl, R² cannot be benzyl; or astereoisomer, enantiomer, racemate, pharmaceutically acceptable salt,solvate, hydrate or polymorph thereof.

In another embodiment, the invention provides a compound of thefollowing formula:

wherein

R¹ is unsubstituted or substituted and selected from C₁-C₁₀ alkyl,C₂-C₁₀ alkenyl, cycloalkyl, aryl, heterocyclyl, heteroaryl, C₁-C₁₀alkyl-C₂-C₁₀ alkenyl, C₁-C₁₀ alkylcycloalkyl, C₁-C₁₀ alkylaryl, C₁-C₁₀alkylheterocyclyl and C₁-C₁₀ alkylheteroaryl;

R² is unsubstituted or substituted and selected from C₁-C₁₀ alkyl,cycloalkyl, heterocyclyl, heteroaryl, C₁-C₁₀ alkylcycloalkyl, C₁-C₁₀alkylheterocyclyl and C₁-C₁₀ alkylheteroaryl;

R³ and R⁴ are independently hydrogen or C₁-C₁₀ alkyl; and

n is 4, 5, 6, or 7;

or a stereoisomer, enantiomer, racemate, pharmaceutically acceptablesalt, solvate, hydrate or polymorph thereof.

In one embodiment, R² is unsubstituted or substituted and selected fromC₁-C₁₀ alkyl, C₁-C₁₀ alkylheterocyclyl and C₁-C₁₀ alkylheteroaryl. Inone embodiment, R² is unsubstituted or substituted and selected fromC₁-C₁₀ alkyl, heterocyclyl, heteroaryl, C₁-C₁₀ alkylcycloalkyl, C₁-C₁₀alkylheterocyclyl and C₁-C₁₀ alkylheteroaryl. In one embodiment, R² isunsubstituted or substituted C₁-C₁₀ alkyl. In another embodiment of theinstant invention, R² is C₁-C₄ alkylheterocyclyl or C₁-C₄alkylheteroaryl. In one embodiment, the heteroaryl group is a 5- to6-membered ring with 1 or 2 heteroatoms. In one embodiment, theheterocyclic group is a 5- to 6-membered ring with 1 or 2 heteroatoms.In one embodiment, the cycloalkyl group is a 5- to 6-membered ring. Saidheterocyclic, heteroaryl, alkyl or cycloalkyl group can be optionallysubstituted with one, two or three substituents R^(sub) as set forthabove. In yet another embodiment of the instant invention, R² in thecompounds of formula I is unsubstituted or substituted and selected fromtert-butyl, (C₀-C₃)alkyl pyridyl, (C₀-C₃)alkyl pyrrolidinyl and(C₀-C₃)alkyl morpholinyl.

In another embodiment, R¹ in the compounds of formula I is phenyl. Inanother embodiment, R² in the compounds of formula I is phenyl. Saidphenyl substituent(s) can be optionally substituted with one, two orthree substituents R^(sub) as set forth above.

In another embodiment, R¹ in the compounds of formula I is thiazolyl. Inanother embodiment, R² in the compounds of formula I is thiazolyl. Saidthiazolyl substituent(s) can be optionally substituted with one, two orthree substituents R^(sub) as set forth above.

In another embodiment, R¹ in the compounds of formula I is tert-butyl.In another embodiment, R² in the compounds of formula I is tert-butyl.Said tert-butyl substituent(s) can be optionally substituted with one,two or three substituents R^(sub) as set forth above.

Thus, in another embodiment, the present invention provides a compoundrepresented by the following structural formula:

wherein

R¹ is thiazolyl or tert-butyl;

R² is unsubstituted or substituted and selected from C₁-C₁₀ alkyl,C₂-C₁₀ alkenyl, cycloalkyl, aryl, heterocyclyl, heteroaryl, C₁-C₁₀alkyl-C₂-C₁₀ alkenyl, C₁-C₁₀ alkylcycloalkyl, C₁-C₁₀ alkylaryl, C₁-C₁₀alkylheterocyclyl and C₁-C₁₀ alkylheteroaryl;

R³ and R⁴ are independently hydrogen or C₁-C₁₀ alkyl; and

n is 4, 5, 6 or 7;

or a stereoisomer, pharmaceutically acceptable salt thereof.

In another embodiment, the present invention provides a compoundrepresented by the following structural formula:

wherein

R¹ is unsubstituted or substituted and selected from C₁-C₁₀ alkyl,C₂-C₁₀ alkenyl, cycloalkyl, aryl, heterocyclyl, heteroaryl, C₁-C₁₀alkyl-C₂-C₁₀ alkenyl, C₁-C₁₀ alkylcycloalkyl, C₁-C₁₀ alkylaryl, C₁-C₁₀alkylheterocyclyl and C₁-C₁₀ alkylheteroaryl;

R² is tert-butyl, phenyl or thiazolyl;

R³ and R⁴ are independently hydrogen or C₁-C₁₀ alkyl; and

n is 4, 5, 6 or 7;

or a stereoisomer, pharmaceutically acceptable salt thereof.

In another embodiment of the instant invention, the compounds areillustrated by the compound of formula IA:

wherein R¹, R², R³ and R⁴ are as defined above for the compound offormula I and wherein n is 5 or 6, or a stereoisomer, enantiomer,racemate, pharmaceutically acceptable salt, solvate, hydrate orpolymorph thereof.

In one embodiment of the instant invention, R¹ and/or R² in thecompounds of formula I are unsubstituted. In another embodiment, R¹and/or R² in the compounds of formula I are substituted with one, two orthree substituents independently selected from R^(sub), wherein R^(sub)is a substitutent selected from C₁-C₁₀ alkyl, C₁-C₁₀ haloalkyl, C₂-C₁₀alkenyl, cycloalkyl, aryl (e.g. phenyl), heterocyclyl, heteroaryl,C₁-C₁₀ alkyl-C₂-C₁₀ alkenyl, C₁-C₁₀ alkylcycloalkyl, C₁-C₁₀ alkylaryl,C₁-C₁₀ alkylheterocyclyl, C₁-C₁₀ alkylheteroaryl, halogen, hydroxy,C₁-C₁₀ alkyloxy, C₁-C₁₀ haloalkyloxy, aryloxy, nitro, oxo, —CN, —C(═O)H,—C(═O)OH, amino, N—C₁-C₁₀ alkylamino, N,N-di C₁-C₁₀ alkylamino,N-arylamino, N,N-diarylamino, N—C₁-C₁₀ alkyl-N-arylamino, azido, andC(═O)OR wherein R is aryl or C₁-C₁₀ alkyl. In another embodiment,R^(sub) in the compounds of formula I is independently selected fromC₁-C₄ alkyl, C₁-C₄ haloalkyl, C₁-C₄ haloalkyloxy, C₁-C₄ alkyloxy, aryl(e.g. phenyl), halogen and nitro. Specific non-limiting examples of suchsubstituents include one, two or three substituents selected from Cl,Br, CF₃, OCH₃, Ph, NO₂, OPh, CH₃, CH(CH₃)₂, C(CH₃)₃ and OCF₃.

In another embodiment of the instant invention, the compounds areillustrated by a compound of Formula II:

wherein

R^(sub) is independently selected from C₁-C₄ alkyl, C₁-C₄ haloalkyl,C₁-C₄ haloalkyloxy, C₁-C₄ alkyloxy, aryl, halogen and nitro;

n is 5 or 6; and

m is selected from 0, 1, 2 or 3;

all other substituents or variables are as defined under Formula I;

or a stereoisomer, enantiomer, racemate, pharmaceutically acceptablesalt, solvate, hydrate or polymorph thereof.

In another embodiment of the instant invention, the compounds areillustrated by a compound of Formula IIA:

wherein R², R³ and R⁴ and n are defined above for the compound offormula II, or a stereoisomer, enantiomer, racemate, pharmaceuticallyacceptable salt, solvate, hydrate or polymorph thereof.

In one embodiment of the instant invention, the phenyl and/or R² in thecompounds of formula II are unsubstituted. In another embodiment, thephenyl and/or R² in the compounds of formula II are substituted withone, two or three substituents independently selected from R^(sub),wherein R^(sub) is a substitutent selected from C₁-C₁₀ alkyl, C₁-C₁₀haloalkyl, C₂-C₁₀ alkenyl, cycloalkyl, aryl (e.g. phenyl), heterocyclyl,heteroaryl, C₁-C₁₀ alkyl-C₂-C₁₀ alkenyl, C₁-C₁₀ alkylcycloalkyl, C₁-C₁₀alkylaryl, C₁-C₁₀ alkylheterocyclyl, C₁-C₁₀ alkylheteroaryl, halogen,hydroxy, C₁-C₁₀ alkyloxy, C₁-C₁₀ haloalkyloxy, aryloxy, nitro, oxo, —CN,—C(═O)H, —C(═O)OH, amino, N—C₁-C₁₀ alkylamino, N,N-di C₁-C₁₀ alkylamino,N-arylamino, N,N-diarylamino, N—C₁-C₁₀ alkyl-N-arylamino, azido, andC(═O)OR wherein R is aryl or C₁-C₁₀ alkyl. In another embodiment,R^(sub) in the compounds of formula II is independently selected fromC₁-C₄ alkyl, C₁-C₄ haloalkyl, C₁-C₄ haloalkyloxy, C₁-C₄ alkyloxy, aryl,halogen and nitro. Specific non-limiting examples of such substituentsinclude one, two or three substituents selected from Cl, Br, CF₃, OCH₃,Ph, NO₂, OPh, CH₃, CH(CH₃)₂, C(CH₃)₃ and OCF₃.

In another embodiment of the instant invention, R² in the compounds offormula II is unsubstituted or substituted and selected from phenyl,fluorenyl, naphthyl, biphenyl, —CH₂CH₂CH₂Ph, cyclopropyl, cyclohexyl,quinolinyl, isoquinolinyl, —CH₂-quinolinyl, —CH₂-isoquinolinyl,thiazolyl, CH(Ph)₂ and C₁-C₁₀ alkyl. In yet another embodiment of theinstant invention, R² in the compounds of formula II is unsubstituted orsubstituted and selected from phenyl, tert-butyl, (C₀-C₃)alkyl pyridyl,(C₀-C₃)alkyl pyrrolidinyl and (C₀-C₃)alkyl morpholinyl.

In another embodiment of the instant invention is a compound representedby the following structural formula:

wherein

R¹ and R² are, independently of each other, unsubstituted or substitutedand selected from phenyl, fluorenyl, biphenyl, —CH₂CH₂Ph, —CH₂CH₂CH₂Ph,cyclopropyl, cyclohexyl, isoquinolinyl, —CH₂-quinolinyl,—CH₂-isoquinolinyl, thiazolyl, CH(Ph)₂ and C₁-C₁₀ alkyl;

R³ and R⁴ are independently hydrogen or C₁-C₁₀ alkyl; and

n is 4, 5, 6, or 7;

or a stereoisomer, pharmaceutically acceptable salt thereof.

In yet another embodiment of the invention is a compound represented bythe following structural formula:

wherein

R¹ is a substituted heteroaryl group, wherein the substituent is aryl;

R² is unsubstituted or substituted and selected from C₁-C₁₀ alkyl,C₂-C₁₀ alkenyl, cycloalkyl, aryl, heterocyclyl, heteroaryl, C₁-C₁₀alkyl-C₂-C₁₀ alkenyl, C₁-C₁₀ alkylcycloalkyl, C₁-C₁₀ alkylaryl, C₁-C₁₀alkylheterocyclyl and C₁-C₁₀ alkylheteroaryl;

R³ and R⁴ are independently hydrogen or C₁-C₁₀ alkyl; and

n is 4, 5, 6, or 7;

or a stereoisomer, pharmaceutically acceptable salt thereof.

In one embodiment, the heteroaryl group of R¹ is a 5- to 6-membered ringwith 1 or 2 heteroatoms. In one embodiment, the aryl substituent of R¹is a 5- to 6-membered ring. In one embodiment, R¹ is phenyl-thiazolyl.Said aryl or heteroaryl group can be optionally substituted with one,two or three substituents R^(sub) as set forth above.

In another embodiment, R³ in the compounds of formula I and/or II ishydrogen. In another embodiment, R³ in the compounds of formula I and/orII is methyl. In another embodiment, R³ in the compounds of formula IAand/or IIA is hydrogen. In another embodiment, R³ in the compounds offormula IA and/or IIA is methyl.

In another embodiment, R⁴ in the compounds of formula I and/or II ishydrogen. In another embodiment, R⁴ in the compounds of formula I and/orII is methyl. In another embodiment, R⁴ in the compounds of formula IAand/or IIA is hydrogen. In another embodiment, R⁴ in the compounds offormula IA and/or IIA is methyl.

In another embodiment, the present invention includes enantiomers of thecompounds of formula I. In yet another embodiment, the present inventionincludes racemates of the compounds of formula I.

Specific examples of the compounds of the instant invention include:

-   (S)-[6-Hydroxycarbamoyl-1-(4-phenyl-thiazol-2-ylcarbamoyl)-hexyl]-carbamic    acid benzyl ester;-   (S)-[6-Hydroxycarbamoyl-1-(4-phenyl-thiazol-2-ylcarbamoyl)-hexyl]-carbamic    acid tert-butyl ester; and-   (S)-(6-Hydroxycarbamoyl-1-phenylcarbamoyl-hexyl)-carbamic acid    tert-butyl ester;-   (6-Hydroxycarbamoyl-1-phenylcarbamoyl-hexyl)-carbamic acid    2-morpholin-4-yl-ethyl ester;-   (6-Hydroxycarbamoyl-1-phenylcarbamoyl-hexyl)-carbamic acid    2-pyridin-2-yl-ethyl ester;-   (6-Hydroxycarbamoyl-1-phenylcarbamoyl-hexyl)-carbamic acid    3-chloro-benzyl ester;-   (6-Hydroxycarbamoyl-1-phenylcarbamoyl-hexyl)-carbamic acid    2-pyrrolidin-1-yl-ethyl ester;    or a stereoisomer, enantiomer, racemate, pharmaceutically acceptable    salt, solvate, hydrate or polymorph thereof.

When any variable (e.g. R¹, R², R³, etc.) occurs more than one time inany constituent, its definition on each occurrence is independent atevery other occurrence. Also, combinations of substituents and variablesare permissible only if such combinations result in stable compounds.Lines drawn into the ring systems from substituents represent that theindicated bond may be attached to any of the substitutable ring atoms.If the ring system is polycyclic, it is intended that the bond beattached to any of the suitable carbon atoms on the proximal ring only.

It is understood that substituents and substitution patterns on thecompounds of the instant invention can be selected by one of ordinaryskill in the art to provide compounds that are chemically stable andthat can be readily synthesized by techniques known in the art, as wellas those methods set forth below, from readily available startingmaterials. If a substituent is itself substituted with more than onegroup, it is understood that these multiple groups may be on the samecarbon or on different carbons, so long as a stable structure results.The phrases “optionally substituted” or “optionally substituted with oneor more substituents” or “unsubstituted or substituted”, used hereininterchangeably, should be taken to be equivalent to the phrase“optionally substituted with at least one substituent” and in such casesin one embodiment will have from zero to three substituents. In oneembodiment, the phrases “optionally substituted” or “optionallysubstituted with one or more substituents” or “unsubstituted orsubstituted” should be taken to mean the phrase “optionally substitutedwith one, two or three substituents”.

As used herein, “alkyl” is intended to include both branched andstraight-chain saturated aliphatic hydrocarbon groups having thespecified number of carbon atoms. For example, C₁-C₁₀, as in “C₁-C₁₀alkyl” is defined to include groups having 1, 2, 3, 4, 5, 6, 7, 8, 9 or10 carbons in a linear or branched arrangement. For example, “C₁-C₁₀alkyl” specifically includes methyl, ethyl, n-propyl, i-propyl, n-butyl,t-butyl, i-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, and so on.The term “cycloalkyl” means a monocyclic saturated aliphatic hydrocarbongroup having the specified number of carbon atoms. For example,“cycloalkyl” includes cyclopropyl, methyl-cyclopropyl,2,2-dimethyl-cyclobutyl, 2-ethyl-cyclopentyl, cyclohexyl, and so on. Inan embodiment of the invention the term “cycloalkyl” includes the groupsdescribed immediately above and further includes monocyclic unsaturatedaliphatic hydrocarbon groups. For example, “cycloalkyl” as defined inthis embodiment includes cyclopropyl, methyl-cyclopropyl,2,2-dimethyl-cyclobutyl, 2-ethyl-cyclopentyl, cyclohexyl, cyclopentenyl,cyclobutenyl and so on.

The term “alkylene” means a hydrocarbon diradical group having thespecified number of carbon atoms. For example, “alkylene” includes—CH₂—, —CH₂CH₂— and the like.

The terms “alkylalkenyl” “alkylcycloalkyl”, alkylaryl”,alkylheterocyclyl” or alkylheteroaryl” mean an alkyl radical linked toan alkenyl, cycloalkyl, aryl, heterocyclyl or heteroaryl group,respectively. For example, in the phrase “C₁-C₆ alkylaryl” or “C₁-C₆alkylheteroaryl” the term “C₁-C₆” refers to the alkyl portion of themoiety and does not describe the number of atoms in the aryl andheteroaryl portion of the moiety.

If no number of carbon atoms is specified, the term “alkenyl” refers toa non-aromatic hydrocarbon radical, straight, branched or cyclic,containing from 2 to 10 carbon atoms and at least one carbon to carbondouble bond. Preferably one carbon to carbon double bond is present, andup to four non-aromatic carbon-carbon double bonds may be present. Thus,“C₂-C₆ alkenyl” means an alkenyl radical having from 2 to 6 carbonatoms. Alkenyl groups include ethenyl, propenyl, butenyl,2-methylbutenyl and cyclohexenyl. The straight, branched or cyclicportion of the alkenyl group may contain double bonds and may besubstituted if a substituted alkenyl group is indicated.

The term “alkynyl” refers to a hydrocarbon radical straight, branched orcyclic, containing from 2 to 10 carbon atoms and at least one carbon tocarbon triple bond. Up to three carbon-carbon triple bonds may bepresent. Thus, “C₂-C₆ alkynyl” means an alkynyl radical having from 2 to6 carbon atoms. Alkynyl groups include ethynyl, propynyl, butynyl,3-methylbutynyl and so on. The straight, branched or cyclic portion ofthe alkynyl group may contain triple bonds and may be substituted if asubstituted alkynyl group is indicated.

In certain instances, substituents may be defined with a range ofcarbons that includes zero, such as (C₀-C₆)alkylene-aryl. If aryl istaken to be phenyl, (C₀-C₆)alkylene-aryl would include phenyl itself aswell as —CH₂Ph, —CH₂CH₂Ph, —CH₂CH₂CH₂Ph, CH(CH₃)CH₂CH(CH₃)Ph, and so on.

As used herein, “aryl” is intended to mean any stable monocyclic orbicyclic carbon ring of up to 7 atoms in each ring, wherein at least onering is aromatic. Examples of such aryl elements include phenyl,naphthyl, fluorenyl, tetrahydronaphthyl, indanyl and biphenyl. In caseswhere the aryl substituent is bicyclic and one ring is non-aromatic, itis understood that attachment is via the aromatic ring.

The term heteroaryl, as used herein, represents a stable monocyclic orbicyclic ring of up to 7 atoms in each ring, wherein at least one ringis aromatic and contains from 1 to 4 heteroatoms selected from the groupconsisting of O, N and S. Heteroaryl groups within the scope of thisdefinition include but are not limited to: acridinyl, carbazolyl,cinnolinyl, quinoxalinyl, pyrrazolyl, indolyl, benzotriazolyl, furanyl,thienyl, benzothienyl, benzofuranyl, quinolinyl, isoquinolinyl,oxazolyl, isoxazolyl, indolyl, pyrazinyl, pyridazinyl, pyridinyl,pyrimidinyl, pyrrolyl, tetrahydroquinoline. As with the definition ofheterocycle below, “heteroaryl” is also understood to include theN-oxide derivative of any nitrogen-containing heteroaryl. In cases wherethe heteroaryl substituent is bicyclic and one ring is non-aromatic orcontains no heteroatoms, it is understood that attachment is via thearomatic ring or via the heteroatom containing ring, respectively.

The term “heterocycle” or “heterocyclyl” as used herein is intended tomean a 3- to 10-membered aromatic or nonaromatic heterocycle containingfrom 1 to 4 heteroatoms selected from the group consisting of O, N andS, and includes bicyclic groups. “Heterocyclyl” therefore includes theabove mentioned heteroaryls, as well as dihydro and tetrathydro analogsthereof. Further examples of “heterocyclyl” include, but are not limitedto the following: azetidinyl, benzoimidazolyl, benzofuranyl,benzofurazanyl, benzopyrazolyl, benzotriazolyl, benzothiophenyl,benzoxazolyl, carbazolyl, carbolinyl, cinnolinyl, furanyl, imidazolyl,indolinyl, indolyl, indolazinyl, indazolyl, isobenzofuranyl, isoindolyl,isoquinolyl, isothiazolyl, isoxazolyl, naphthpyridinyl, oxadiazolyl,oxazolyl, oxazoline, isoxazoline, oxetanyl, pyranyl, pyrazinyl,pyrazolyl, pyridazinyl, pyridopyridinyl, pyridazinyl, pyridyl,pyrimidyl, pyrrolyl, quinazolinyl, quinolyl, quinoxalinyl,tetrahydropyranyl, tetrahydrothiopyranyl, tetrahydroisoquinolinyl,tetrazolyl, tetrazolopyridyl, thiadiazolyl, thiazolyl, thienyl,triazolyl, azetidinyl, 1,4-dioxanyl, hexahydroazepinyl, piperazinyl,piperidinyl, pyridin-2-onyl, pyrrolidinyl, morpholinyl, thiomorpholinyl,dihydrobenzoimidazolyl, dihydrobenzofuranyl, dihydrobenzothiophenyl,dihydrobenzoxazolyl, dihydrofuranyl, dihydroimidazolyl, dihydroindolyl,dihydroisooxazolyl, dihydroisothiazolyl, dihydrooxadiazolyl,dihydrooxazolyl, dihydropyrazinyl, dihydropyrazolyl, dihydropyridinyl,dihydropyrimidinyl, dihydropyrrolyl, dihydroquinolinyl,dihydrotetrazolyl, dihydrothiadiazolyl, dihydrothiazolyl,dihydrothienyl, dihydrotriazolyl, dihydroazetidinyl,methylenedioxybenzoyl, tetrahydrofuranyl, and tetrahydrothienyl, andN-oxides thereof. Attachment of a heterocyclyl substituent can occur viaa carbon atom or via a heteroatom.

In an embodiment, the term “heterocycle” or “heterocyclyl” as usedherein is intended to mean a 5- to 10-membered aromatic or nonaromaticheterocycle containing from 1 to 4 heteroatoms selected from the groupconsisting of O, N and S, and includes bicyclic groups. “Heterocyclyl”in this embodiment therefore includes the above mentioned heteroaryls,as well as dihydro and tetrahydro analogs thereof. Further examples of“heterocyclyl” include, but are not limited to the following:benzoimidazolyl, benzofuranyl, benzofurazanyl, benzopyrazolyl,benzotriazolyl, benzothiophenyl, benzoxazolyl, carbazolyl, carbolinyl,cinnolinyl, furanyl, imidazolyl, indolinyl, indolyl, indolazinyl,indazolyl, isobenzofuranyl, isoindolyl, isoquinolyl, isothiazolyl,isoxazolyl, naphthpyridinyl, oxadiazolyl, oxazolyl, oxazoline,isoxazoline, oxetanyl, pyranyl, pyrazinyl, pyrazolyl, pyridazinyl,pyridopyridinyl, pyridazinyl, pyridyl, pyrimidyl, pyrrolyl,quinazolinyl, quinolyl, quinoxalinyl, tetrahydropyranyl,tetrahydrothiopyranyl, tetrahydroisoquinolinyl, tetrazolyl,tetrazolopyridyl, thiadiazolyl, thiazolyl, thienyl, triazolyl,azetidinyl, 1,4-dioxanyl, hexahydroazepinyl, piperazinyl, piperidinyl,pyridin-2-onyl, pyrrolidinyl, morpholinyl, thiomorpholinyl,dihydrobenzoimidazolyl, dihydrobenzofuranyl, dihydrobenzothiophenyl,dihydrobenzoxazolyl, dihydrofuranyl, dihydroimidazolyl, dihydroindolyl,dihydroisooxazolyl, dihydroisothiazolyl, dihydrooxadiazolyl,dihydrooxazolyl, dihydropyrazinyl, dihydropyrazolyl, dihydropyridinyl,dihydropyrimidinyl, dihydropyrrolyl, dihydroquinolinyl,dihydrotetrazolyl, dihydrothiadiazolyl, dihydrothiazolyl,dihydrothienyl, dihydrotriazolyl, dihydroazetidinyl,methylenedioxybenzoyl, tetrahydrofuranyl, and tetrahydrothienyl, andN-oxides thereof. Attachment of a heterocyclyl substituent can occur viaa carbon atom or via a heteroatom.

In another embodiment, heterocycle is selected from quinolinyl,isoquinolinyl, and thiazolyl.

As appreciated by those of skill in the art, “halo” or “halogen” as usedherein is intended to include chloro, fluoro, bromo and iodo.

A “haloalkyl group” is an alkyl group of indicated number of carbonatoms, that is attached to one or more halogen atoms (e.g. one two,three, four, five or six halogen atoms), including chloro, fluoro, bromoand iodo. An example of a haloalkyl group is CF₃.

An “aryloxy group” is an aryl group that is attached to a compound viaan oxygen (e.g., phenoxy).

“Alkoxy” (alkyloxy) represents either a cyclic or non-cyclic alkyl groupof indicated number of carbon atoms attached through an oxygen bridge.“Alkoxy” or “alkyloxy” therefore encompasses the definitions of alkyland cycloalkyl above. An example of an alkoxy group is OCH₃.

“Haloalkoxy” (haloalkyloxy”) represents either a cyclic ornon-cyclic-alkyl group of indicated number of carbon atoms attachedthrough an oxygen bridge, which groups is attached to at least onehalogen atom (e.g. one two, three, four, five or six halogen atoms),including chloro, fluoro, bromo and iodo. An example of a haloalkyloxygroup is OCF₃.

An “arylalkoxy group” (arylalkyloxy) represents an arylalkyl group thatis attached to a compound via an oxygen on the alkyl portion of thearylalkyl (e.g., phenylmethoxy).

An “arylamino group”, represents an aryl group that is attached to acompound via a nitrogen.

As used herein, an “arylalkylamino group” is an arylalkyl group that isattached to a compound via a nitrogen on the alkyl portion of thearylalkyl.

As used herein, many moieties or groups are referred to as being either“substituted or unsubstituted” or “optionally substituted”. When amoiety is referred to as substituted, it denotes that any portion of themoiety that is known to one skilled in the art as being available forsubstitution can be substituted. The phrase “optionally substituted withone or more substituents” means, in one embodiment, “zero to fivesubstituents”, and in other embodiments, one substituent, twosubstituents, three substituents, four substituents or fivesubstituents. For example, the substitutable group can be a hydrogenatom that is replaced with a group other than hydrogen (i.e., asubstituent group). Multiple substituent groups can be present. Whenmultiple substituents are present, the substituents can be the same ordifferent and substitution can be at any of the substitutable sites.Such means for substitution are well known in the art. For purposes ofexemplification, which should not be construed as limiting the scope ofthis invention, some examples of groups that are substituents(designated herein as “R^(sub)”) are: C₁-C₁₀ alkyl groups, e.g. CH₃,CH(CH₃)₂, C(CH₃)₃ etc (which can also be substituted with one or moresubstituents); C₁-C₁₀ haloalkyl groups, e.g. CF₃, (which can also besubstituted with one or more substituents); C₁-C₁₀ alkyloxy groups, e.g.OCH₃, (which can be substituted); C₁-C₁₀ haloalkyloxy groups, e.g. OCF₃;a halogen or halo group (F, Cl, Br, I); hydroxyl; nitro; oxo; —CN; —COH;—COOH; amino; azido; N-alkylamino or N,N-dialkylamino (in which thealkyl groups can also be substituted); N-arylamino or N,N-diarylamino(in which the aryl groups can also be substituted); aryl (e.g. phenyl),esters (—C(O)—OR, where R can be a group such as alkyl, aryl, etc.,which can be substituted); aryl (which can be substituted); cycloalkyl(which can be substituted) alkylaryl (which can be substituted);alkylheterocyclyl (which can be substituted); alkylcycloalkyl (which canbe substituted), and aryloxy (e.g. —OPh).

Included in the instant invention is the free form of compounds ofFormula I, as well as the pharmaceutically acceptable salts andstereoisomers thereof. Some of the specific compounds exemplified hereinare the protonated salts of amine compounds. The term “free form” refersto the amine compounds in non-salt form. The encompassedpharmaceutically acceptable salts not only include the salts exemplifiedfor the specific compounds described herein, but also all the typicalpharmaceutically acceptable salts of the free form of compounds ofFormula I. The free form of the specific salt compounds described may beisolated using techniques known in the art. For example, the free formmay be regenerated by treating the salt with a suitable dilute aqueousbase solution such as dilute aqueous NaOH, potassium carbonate, ammoniaand sodium bicarbonate. The free forms may differ from their respectivesalt forms somewhat in certain physical properties, such as solubilityin polar solvents, but the acid and base salts are otherwisepharmaceutically equivalent to their respective free forms for purposesof the invention.

Many organic compounds exist in optically active forms having theability to rotate the plane of plane-polarized light. In describing anoptically active compound, the prefixes D and L or R and S are used todenote the absolute configuration of the molecule about its chiralcenter(s). The prefixes d and 1 or (+) and (−) are employed to designatethe sign of rotation of plane-polarized light by the compound, with (−)or meaning that the compound is levorotatory. A compound prefixed with(+) or d is dextrorotatory. For a given chemical structure, thesecompounds, called stereoisomers, are identical except that they arenon-superimposable mirror images of one another. A specific stereoisomercan also be referred to as an enantiomer, and a mixture of such isomersis often called an enantiomeric mixture. A 50:50 mixture of enantiomersis referred to as a racemic mixture. Many of the compounds describedherein can have one or more chiral centers and therefore can exist indifferent enantiomeric forms. If desired, a chiral carbon can bedesignated with an asterisk (*). When bonds to the chiral carbon aredepicted as straight lines in the formulas of the invention, it isunderstood that both the (R) and (S) configurations of the chiralcarbon, and hence both enantiomers and mixtures thereof, are embracedwithin the formula. As is used in the art, when it is desired to specifythe absolute configuration about a chiral carbon, one of the bonds tothe chiral carbon can be depicted as a wedge (bonds to atoms above theplane) and the other can be depicted as a series or wedge of shortparallel lines is (bonds to atoms below the plane). TheCahn-Inglod-Prelog system can be used to assign the (R) or (S)configuration to a chiral carbon.

When the HDAC inhibitors of the present invention contain one chiralcenter, the compounds exist in two enantiomeric forms and the presentinvention includes both enantiomers and mixtures of enantiomers, such asthe specific 50:50 mixture referred to as a racemic mixtures. Theenantiomers can be resolved by methods known to those skilled in theart, such as formation of diastereoisomeric salts which may beseparated, for example, by crystallization (see, CRC Handbook of OpticalResolutions via Diastereomeric Salt Formation by David Kozma (CRC Press,2001)); formation of diastereoisomeric derivatives or complexes whichmay be separated, for example, by crystallization, gas-liquid or liquidchromatography; selective reaction of one enantiomer with anenantiomer-specific reagent, for example enzymatic esterification; orgas-liquid or liquid chromatography in a chiral environment, for exampleon a chiral support for example silica with a bound chiral ligand or inthe presence of a chiral solvent. It will be appreciated that where thedesired enantiomer is converted into another chemical entity by one ofthe separation procedures described above, a further step is required toliberate the desired enantiomeric form. Alternatively, specificenantiomers may be synthesized by asymmetric synthesis using opticallyactive reagents, substrates, catalysts or solvents, or by converting oneenantiomer into the other by asymmetric transformation.

Designation of a specific absolute configuration at a chiral carbon ofthe compounds of the invention is understood to mean that the designatedenantiomeric form of the compounds is in enantiomeric excess (ee) or inother words is substantially free from the other enantiomer. Forexample, the “R” forms of the compounds are substantially free from the“S” forms of the compounds and are, thus, in enantiomeric excess of the“S” forms. Conversely, “S” forms of the compounds are substantially freeof “R” forms of the compounds and are, thus, in enantiomeric excess ofthe “R” forms. Enantiomeric excess, as used herein, is the presence of aparticular enantiomer at greater than 50%. For example, the enantiomericexcess can be about 60% or more, such as about 70% or more, for exampleabout 80% or more, such as about 90% or more. In a particular embodimentwhen a specific absolute configuration is designated, the enantiomericexcess of depicted compounds is at least about 90%. In a more particularembodiment, the enantiomeric excess of the compounds is at least about95%, such as at least about 97.5%, for example, at least 99%enantiomeric excess.

When a compound of the present invention has two or more chiral carbonsit can have more than two optical isomers and can exist indiastereoisomeric forms. For example, when there are two chiral carbons,the compound can have up to 4 optical isomers and 2 pairs of enantiomers((S,S)/(R,R) and (R,S)/(S,R)). The pairs of enantiomers (e.g.,(S,S)/(R,R)) are mirror image stereoisomers of one another. Thestereoisomers that are not mirror-images (e.g., (S,S) and (R,S)) arediastereomers. The diastereoisomeric pairs may be separated by methodsknown to those skilled in the art, for example chromatography orcrystallization and the individual enantiomers within each pair may beseparated as described above. The present invention includes eachdiastereoisomer of such compounds and mixtures thereof.

The hydroxamic acid derivatives described herein can, as noted above, beprepared in the form of their pharmaceutically acceptable salts.

The pharmaceutically acceptable salts of the instant compounds can besynthesized from the compounds of this invention which contain a basicor acidic moiety by conventional chemical methods. Generally, the saltsof the basic compounds are prepared either by ion exchangechromatography or by reacting the free base with stoichiometric amountsor with an excess of the desired salt-forming inorganic or organic acidin a suitable solvent or various combinations of solvents. Similarly,the salts of the acidic compounds are formed by reactions with theappropriate inorganic or organic base.

Thus, pharmaceutically acceptable salts of the compounds of thisinvention include the conventional non-toxic salts of the compounds ofthis invention as formed by reacting a basic instant compound with aninorganic or organic acid. For example, conventional non-toxic saltsinclude those derived from inorganic acids such as hydrochloric,hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like, aswell as salts prepared from organic acids such as acetic, propionic,succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic,pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic,salicylic, sulfanilic, 2-acetoxy-benzoic, fumaric, toluenesulfonic,methanesulfonic, ethane disulfonic, oxalic, isethionic, trifluoroaceticand the like.

When the compound of the present invention is acidic, suitable“pharmaceutically acceptable salts” refers to salts prepared formpharmaceutically acceptable non-toxic bases including inorganic basesand organic bases. Salts derived from inorganic bases include aluminum,ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganicsalts, manganous, potassium, sodium, zinc and the like. Particularlypreferred are the ammonium, calcium, magnesium, potassium and sodiumsalts. Salts derived from pharmaceutically acceptable organic non-toxicbases include salts of primary, secondary and tertiary amines,substituted amines including naturally occurring substituted amines,cyclic amines and basic ion exchange resins, such as arginine, betainecaffeine, choline, N,N¹-dibenzylethylenediamine, diethylamin,2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine,ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine,glucosamine, histidine, hydrabamine, isopropylamine, lysine,methylglucamine, morpholine, piperazine, piperidine, polyamine resins,procaine, purines, theobromine, triethylamine, trimethylaminetripropylamine, tromethamine and the like.

The preparation of the pharmaceutically acceptable salts described aboveand other typical pharmaceutically acceptable salts is more fullydescribed by Berg et al., “Pharmaceutical Salts,” J. Pharm. Sci.,1977:66:1-19.

It will also be noted that the compounds of the present invention arepotentially internal salts or zwitterions, since under physiologicalconditions a deprotonated acidic moiety in the compound, such as acarboxyl group, may be anionic, and this electronic charge might then bebalanced off internally against the cationic charge of a protonated oralkylated basic moiety, such as a quaternary nitrogen atom.

The active compounds disclosed can, as noted above, also be prepared inthe form of their hydrates. The term “hydrate” includes but is notlimited to hemihydrate, monohydrate, dihydrate, trihydrate, tetrahydrateand the like.

The active compounds disclosed can, as noted above, also be prepared inthe form of a solvate with any organic or inorganic solvent, for examplealcohols such as methanol, ethanol, propanol and isopropanol, ketonessuch as acetone, aromatic solvents and the like.

The active compounds disclosed can also be prepared in any solid orliquid physical form. For example, the compound can be in a crystallineform, in amorphous form, and have any particle size. Furthermore, thecompound particles may be micronized, or may be agglomerated,particulate granules, powders, oils, oily suspensions or any other formof solid or liquid physical form.

The compounds of the present invention may also exhibit polymorphism.This invention further includes different polymorphs of the compounds ofthe present invention. The term “polymorph” refers to a particularcrystalline state of a substance, having particular physical propertiessuch as X-ray diffraction, IR spectra, melting point, and the like.

This invention is also intended to encompass pro-drugs of the hydroxamicacid derivatives disclosed herein. A prodrug of any of the compounds canbe made using well known pharmacological techniques.

This invention, in addition to the above listed compounds, is intendedto encompass the use of homologs and analogs of such compounds. In thiscontext, homologs are molecules having substantial structuralsimilarities to the above-described compounds and analogs are moleculeshaving substantial biological similarities regardless of structuralsimilarities.

The compounds of this invention may be prepared by employing reactionsas shown in the following schemes, in addition to other standardmanipulations that are known in the literature or exemplified in theexperimental procedures. The illustrative schemes below, therefore, arenot limited by the compounds listed or by any particular substituentsemployed for illustrative purposes. Substituent numbering as shown inthe schemes does not necessarily correlate to that used in the claimsand often, for clarity, a single substituent is shown attached to thecompound where multiple substituents are allowed under the definitionsof Formula I hereinabove.

Schemes

Scheme 1 illustrates the synthesis of the use of the free amine ofaminosuberic acid to generate the carbamate derivatives of the presentinvention.

Utility

The invention also relates to methods of using the hydroxamic acidderivatives described herein. As demonstrated herein, the hydroxamicacid derivatives of the present invention are useful for the treatmentof cancer. In addition, there is a wide range of other diseases forwhich hydroxamic acid derivatives have been found useful. Non-limitingexamples are thioredoxin (TRX)-mediated diseases as described herein,diseases of the central nervous system (CNS) as described herein, andtreatment of restenosis by providing a stent device comprising thehydroxamic acid derivatives as described herein.

As demonstrated herein, the hydroxamic acid derivatives of the presentinvention are useful for the treatment of cancer.

Accordingly, in one embodiment, the instant invention relates to amethod of treating cancer in a subject in need of treatment comprisingadministering to said subject a therapeutically effective amount of thehydroxamic acid derivatives described herein.

In another embodiment, the instant invention relates to the use of anyone or more of the hydroxamic acid derivatives disclosed herein in thepreparation of a medicament. In another embodiment, the instantinvention relates to the use of any one or more of the hydroxamic acidderivatives disclosed herein in the preparation of a medicament for thetreatment of cancer in a subject in need of such treatment.

The compounds, compositions and methods provided herein are particularlydeemed useful for the treatment of cancer including solid tumors such asskin, breast, brain, cervical carcinomas, testicular carcinomas, etc.

The term “cancer” refers to any cancer caused by the proliferation ofneoplastic cells, such as solid tumors, neoplasms, carcinomas, sarcomas,leukemias, lymphomas and the like. For example, cancers include, but arenot limited to: leukemias including acute leukemias and chronicleukemias such as acute lymphocytic leukemia (ALL), Acute myeloidleukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenousleukemia (CML) and Hairy Cell Leukemia; lymphomas such as cutaneousT-cell lymphomas (CTCL), noncutaneous peripheral T-cell lymphomas,lymphomas associated with human T-cell lymphotrophic virus (HTLV) suchas adult T-cell leukemia/lymphoma (ATLL), Hodgkin's disease andnon-Hodgkin's lymphomas, large-cell lymphomas, diffuse large B-celllymphoma (DLBCL); Burkitt's lymphoma; primary central nervous system(CNS) lymphoma; multiple myeloma; childhood solid tumors such as braintumors, neuroblastoma, retinoblastoma, Wilm's tumor, bone tumors, andsoft-tissue sarcomas, common solid tumors of adults such as head andneck cancers (e.g., oral, laryngeal and esophageal), genito urinarycancers (e.g., prostate, bladder, renal, uterine, ovarian, testicular,rectal and colon), lung cancer, breast cancer.

In a further embodiment, cancers that may be treated by the compounds,compositions and methods of the invention include, but are not limitedto: Cardiac: sarcoma (angiosarcoma, fibrosarcoma, rhabdomyosarcoma,liposarcoma), myxoma, rhabdomyoma, fibroma, lipoma and teratoma; Lung:bronchogenic carcinoma (squamous cell, undifferentiated small cell,undifferentiated large cell, adenocarcinoma), alveolar (bronchiolar)carcinoma, bronchial adenoma, sarcoma, lymphoma, chondromatoushamartoma, mesothelioma; Gastrointestinal: esophagus (squamous cellcarcinoma, adenocarcinoma, leiomyosarcoma, lymphoma), stomach(carcinoma, lymphoma, leiomyosarcoma), pancreas (ductal adenocarcinoma,insulinoma, glucagonoma, gastrinoma, carcinoid tumors, vipoma), smallbowel (adenocarcinoma, lymphoma, carcinoid tumors, Karposi's sarcoma,leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), large bowel(adenocarcinoma, tubular adenoma, villous adenoma, hamartoma,leiomyoma); Genitourinary tract: kidney (adenocarcinoma, Wilm's tumor[nephroblastoma], lymphoma, leukemia), bladder and urethra (squamouscell carcinoma, transitional cell carcinoma, adenocarcinoma), prostate(adenocarcinoma, sarcoma), testis (seminoma, teratoma, embryonalcarcinoma, teratocarcinoma, choriocarcinoma, sarcoma, interstitial cellcarcinoma, fibroma, fibroadenoma, adenomatoid tumors, lipoma); Liver:hepatoma (hepatocellular carcinoma), cholangiocarcinoma, hepatoblastoma,angiosarcoma, hepatocellular adenoma, hemangioma; Bone: osteogenicsarcoma (osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma,chondrosarcoma, Ewing's sarcoma, malignant lymphoma (reticulum cellsarcoma), multiple myeloma, malignant giant cell tumor chordoma,osteochronfroma (osteocartilaginous exostoses), benign chondroma,chondroblastoma, chondromyxofibroma, osteoid osteoma and giant celltumors; Nervous system: skull (osteoma, hemangioma, granuloma, xanthoma,osteitis deformans), meninges (meningioma, meningiosarcoma,gliomatosis), brain (astrocytoma, medulloblastoma, glioma, ependymoma,germinoma [pinealoma], glioblastoma multiform, oligodendroglioma,schwannoma, retinoblastoma, congenital tumors), spinal cordneurofibroma, meningioma, glioma, sarcoma); Gynecological: uterus(endometrial carcinoma), cervix (cervical carcinoma, pre-tumor cervicaldysplasia), ovaries (ovarian carcinoma [serous cystadenocarcinoma,mucinous cystadenocarcinoma, unclassified carcinoma], granulosa-thecalcell tumors, Sertoli-Leydig cell tumors, dysgerminoma, malignantteratoma), vulva (squamous cell carcinoma, intraepithelial carcinoma,adenocarcinoma, fibrosarcoma, melanoma), vagina (clear cell carcinoma,squamous cell carcinoma, botryoid sarcoma (embryonal rhabdomyosarcoma),fallopian tubes (carcinoma); Hematologic: blood (myeloid leukemia [acuteand chronic], acute lymphoblastic leukemia, chronic lymphocyticleukemia, myeloproliferative diseases, multiple myeloma, myelodysplasticsyndrome), Hodgkin's disease, non-Hodgkin's lymphoma [malignantlymphoma]; Skin: malignant melanoma, basal cell carcinoma, squamous cellcarcinoma, Karposi's sarcoma, moles dysplastic nevi, lipoma, angioma,dermatofibroma, keloids, psoriasis; and Adrenal glands: neuroblastoma.Thus, the term “cancerous cell” as provided herein, includes a cellafflicted by any one of the above-identified conditions.

As demonstrated herein, the hydroxamic acid derivatives of the presentinvention are useful for the treatment of a thioredoxin (TRX)-mediateddiseases or disorders.

Accordingly, in one embodiment, the instant invention relates to amethod of treating a thioredoxin (TRX)-mediated disease or disorder in asubject in need of treatment comprising administering to said subject atherapeutically effective amount of the hydroxamic acid derivativesdescribed herein.

In another embodiment, the instant invention relates to the use of anyone or more of the hydroxamic acid derivatives disclosed herein in thepreparation of a medicament for the treatment of a thioredoxin(TRX)-mediated disease or disorder in a subject in need of suchtreatment. Examples of TRX-mediated diseases include, but are notlimited to, acute and chronic inflammatory diseases, autoimmunediseases, allergic diseases, diseases associated with oxidative stress,and diseases characterized by cellular hyperproliferation.

Non-limiting examples are inflammatory conditions of a joint includingrheumatoid arthritis (RA) and psoriatic arthritis; inflammatory boweldiseases such as Crohn's disease and ulcerative colitis;spondyloarthropathies; scleroderma; psoriasis (including T-cell mediatedpsoriasis) and inflammatory dermatoses such an dermatitis, eczema,atopic dermatitis, allergic contact dermatitis, urticaria; vasculitis(e.g., necrotizing, cutaneous, and hypersensitivity vasculitis);eosinphilic myositis, eosinophilic fasciitis; cancers with leukocyteinfiltration of the skin or organs, ischemic injury, including cerebralischemia (e.g., brain injury as a result of trauma, epilepsy, hemorrhageor stroke, each of which may lead to neurodegeneration); HIV, heartfailure, chronic, acute or malignant liver disease, autoimmunethyroiditis; systemic lupus erythematosus, Sjorgren's syndrome, lungdiseases (e.g., ARDS); acute pancreatitis; amyotrophic lateral sclerosis(ALS); Alzheimer's disease; cachexia/anorexia; asthma; atherosclerosis;chronic fatigue syndrome, fever; diabetes (e.g., insulin diabetes orjuvenile onset diabetes); glomerulonephritis; graft versus hostrejection (e.g., in transplantation); hemohorragic shock; hyperalgesia:inflammatory bowel disease; multiple sclerosis; myopathies (e.g., muscleprotein metabolism, esp. in sepsis); osteoporosis; Parkinson's disease;pain; pre-term labor; psoriasis; reperfusion injury; cytokine-inducedtoxicity (e.g., septic shock, endotoxic shock); side effects fromradiation therapy, temporal mandibular joint disease, tumor metastasis;or an inflammatory condition resulting from strain, sprain, cartilagedamage, trauma such as burn, orthopedic surgery, infection or otherdisease processes. Allergic diseases and conditions, include but are notlimited to respiratory allergic diseases such as asthma, allergicrhinitis, hypersensitivity lung diseases, hypersensitivity pneumonitis,eosinophilic pneumonias (e.g., Loeffler's syndrome, chronic eosinophilicpneumonia), delayed-type hypersensitivity, interstitial lung diseases(ILD) (e.g., idiopathic pulmonary fibrosis, or ILD associated withrheumatoid arthritis, systemic lupus erythematosus, ankylosingspondylitis, systemic sclerosis, Sjogren's syndrome, polymyositis ordermatomyositis); systemic anaphylaxis or hypersensitivity responses,drug allergies (e.g., to penicillin, cephalosporins), insect stingallergies, and the like.

As demonstrated herein, the hydroxamic acid derivatives of the presentinvention are useful for the treatment of diseases of the centralnervous system (CNS).

Accordingly, in one embodiment, the instant invention relates to amethod of treating a disease of the central nervous system (CNS) in asubject in need of treatment comprising administering to said subject atherapeutically effective amount of the hydroxamic acid derivativesdescribed herein.

In another embodiment, the instant invention relates to the use of anyone or more of the hydroxamic acid derivatives disclosed herein in thepreparation of a medicament for the treatment of a disease of thecentral nervous system (CNS) in a subject in need of such treatment.

In a particular embodiment, the CNS disease is a neurodegenerativedisease. In a further embodiment, the neurodegenerative disease is aninherited neurodegenerative disease, such as those inheritedneurodegenerative diseases that are polyglutamine expansion diseases.Generally, neurodegenerative diseases can be grouped as follows:

I. Disorders characterized by progressive dementia in the absence ofother prominent neurologic signs, such as Alzheimer's disease; Seniledementia of the Alzheimer type; and Pick's disease (lobar atrophy).

II. Syndromes combining progressive dementia with other prominentneurologic abnormalities such as A) syndromes appearing mainly in adults(e.g., Huntington's disease, Multiple system atrophy combining dementiawith ataxia and/or manifestations of Parkinson's disease, Progressivesupranuclear palsy (Steel-Richardson-Olszewski), diffuse Lewy bodydisease, and corticodentatonigral degeneration); and B) syndromesappearing mainly in children or young adults (e.g., Hallervorden-Spatzdisease and progressive familial myoclonic epilepsy).III. Syndromes of gradually developing abnormalities of posture andmovement such as paralysis agitans (Parkinson's disease), striatonigraldegeneration, progressive supranuclear palsy, torsion dystonia (torsionspasm; dystonia musculorum deformans), spasmodic torticollis and otherdyskinesis, familial tremor, and Gilles de la Tourette syndrome.IV. Syndromes of progressive ataxia such as cerebellar degenerations(e.g., cerebellar cortical degeneration and olivopontocerebellar atrophy(OPCA)); and spinocerebellar degeneration (Friedreich's atazia andrelated disorders).V. Syndrome of central autonomic nervous system failure (Shy-Dragersyndrome).VI. Syndromes of muscular weakness and wasting without sensory changes(motorneuron disease such as amyotrophic lateral sclerosis, spinalmuscular atrophy (e.g., infantile spinal muscular atrophy(Werdnig-Hoffman), juvenile spinal muscular atrophy(Wohlfart-Kugelberg-Welander) and other forms of familial spinalmuscular atrophy), primary lateral sclerosis, and hereditary spasticparaplegia.VII. Syndromes combining muscular weakness and wasting with sensorychanges (progressive neural muscular atrophy; chronic familialpolyneuropathies) such as peroneal muscular atrophy(Charcot-Marie-Tooth), hypertrophic interstitial polyneuropathy(Dejerine-Sottas), and miscellaneous forms of chronic progressiveneuropathy.VIII. Syndromes of progressive visual loss such as pigmentarydegeneration of the retina (retinitis pigmentosa), and hereditary opticatrophy (Leber's disease).

The compounds of the present invention are also useful in the inhibitionof smooth muscle cell proliferation and/or migration and are thus usefulin the prevention and/or treatment of restenosis, for example afterangioplasty and/or stent implantation.

In one embodiment, smooth muscle cell proliferation and/or migration isinhibited and restenosis is prevented and/or treated by providing astent device having one or more of the compounds of the instantinvention in or on the stent device, e.g. coated onto the stent device.The stent device is designed to controllably release the compounds ofthe invention, thereby inhibiting smooth miscle cell proliferationand/or migration and preventing and/or treating restenosis.

Stenosis and restenosis are conditions associated with a narrowing ofblood vessels. Stenosis of blood vessels generally occurs gradually overtime. Restenosis, in contrast, relates to a narrowing of blood vesselsfollowing an endovascular procedure, such as balloon angioplasty and/orstent implantation, or a vascular injury.

Balloon angioplasty is typically performed to open a stenotic bloodvessel; stenting is usually performed to maintain the patency of a bloodvessel after, or in combination with, balloon angioplasty. A stenoticblood vessel is opened with balloon angioplasty by navigating aballoon-tipped catheter to the site of stenosis, and expanding theballoon tip effectively to dilate the occluded blood vessel. In aneffort to maintain the patency of the dilated blood vessel, a stent maybe implanted in the blood vessel to provide intravascular support to theopened section of the blood vessel, thereby limiting the extent to whichthe blood vessel will return to its occluded state after release of theballoon catheter. Restenosis is typically caused by trauma inflictedduring angioplasty, effected by, for example, balloon dilation,atherectomy or laser ablation treatment of the artery. For theseprocedures, restenosis occurs at a rate of about 30% to about 60%depending on the vessel location, lesion length and a number of othervariables. This reduces the overall success of the relativelynon-invasive balloon angioplasty and stenting procedures.

Restenosis is attributed to many factors, including proliferation ofsmooth muscle cells (SMC). SMC proliferation is triggered by the initialmechanical injury to the intima that is sustained at the time of balloonangioplasty and stent implantation. The process is characterized byearly platelet activation and thrombus formation, followed by SMCrecruitment and migration, and, finally, cellular proliferation andextracellular matrix accumulation. Damaged endothelial cells, SMCs,platelets, and macrophages secrete cytokines and growth factors whichpromote restenosis. SMC proliferation represents the final commonpathway leading to neointimal hyperplasia. Therefore, anti-proliferativetherapies aimed at inhibiting specific regulatory events in the cellcycle may constitute the most reasonable approach to restenosis afterangioplasty.

The compounds of the instant invention are, as demonstrated herein,potent HDAC inhibitors that show great potential in the treatment ofcell proliferative diseases or conditions. In addition, the compounds ofthe instant invention are potent inhibitors of SMC proliferation. Thus,the controllable release of the compounds of the invention (e.g. from astent device), is highly advantageous in the inhibition of SMCproliferation and hence in the prevention and/or treatment ofrestenosis.

Thus, in one embodiment, this invention provides a stent devicecomprising one or more of the HDAC inhibitors of the instant invention.The stent device includes a stent body and one or more of the compoundsof the invention provided on or in the stent body. In one embodiment,the compounds are included in a delivery depot which is located on or inthe stent body. In another embodiment, the compounds are coated onto thestent body.

In another embodiment, this invention provides a stent device asdescribed herein for use in inhibiting proliferation and/or migration ofsmooth muscle cells.

In another embodiment, this invention relates to the use of the stentdevice described herein for inhibiting and/or prevention restenosis in asubject in need thereof.

In another embodiment, this invention relates to the use of thecompounds of the instant invention for inhibiting proliferation and/ormigration of non-neoplastic smooth muscle cells in a subject.

In another embodiment, this invention relates to the use of thecompounds of the instant invention for inhibiting and/or preventionrestenosis in a subject in need thereof.

In another embodiment, this invention relates to a method for inhibitingproliferation and/or migration of non-neoplastic smooth muscle cells ina subject, comprising administering to the subject a compound of thepresent invention, in an amount effective to inhibit proliferation ofsmooth muscle cells in the subject.

In another embodiment, this invention relates to a method for preventingor treating restenosis after angioplasty or stent implantation in asubject, comprising administering to the subject an amount of a compoundof the instant invention, effective to prevent restenosis in thesubject.

In another embodiment, this invention relates to a method for preventingor treating restenosis after angioplasty or stent implantation in asubject, comprising positioning a stent device within a lumen of a bloodvessel of the subject, the stent device comprising a stent body and oneor more of the compounds of the present invention provided on or in thestent body. In one embodiment, the compounds are included in a deliverydepot which is located on or in the stent body. In another embodiment,the compounds are coated onto the stent body.

In yet another embodiment, the present invention provides a kitcomprising a stent device as described herein, and a delivery cathetercapable of positioning the stent device within a lumen of a blood vesselof a subject.

Stents are known in the art, and are typically metallic or polymericdevices that are permanently implanted in an expanded form in coronaryand peripheral vessels. Stents can typically by made from a metal, suchas stainless steel, tantalum, titanium alloy, cobalt alloy, silicones ora polymer such as thermoplastic elastomers including polyolefinelastomers and polyamide elastomers or any combinations thereof. A stentis typically inserted by a catheter into a vascular lumen and expandedinto contact with the arterial wall, thereby providing internal supportfor the lumen. Examples of stents are disclosed in U.S. Pat. Nos.4,733,665, 4,800,882 and 4,886,062.

Stents containing drug delivery systems are also known in the art. Forexample, U.S. Pat. Nos. 6,273,913, 6,383,215, 6,238,121, 6,231,600,5,837,008, 5,824,048, and 5,679,400 all teach stents coated with variouspharmaceutical agents. Methods of coating pharmaceutical drugs ontostents are also known to a person of skill in the art.

The term “treating” in its various grammatical forms in relation to thepresent invention refers to preventing (i.e., chemoprevention), curing,reversing, attenuating, alleviating, minimizing, suppressing or haltingthe deleterious effects of a disease state, disease progression, diseasecausative agent (e.g., bacteria or viruses) or other abnormal condition.For example, treatment may involve alleviating a symptom (i.e., notnecessary all symptoms) of a disease or attenuating the progression of adisease. Because some of the inventive methods involve the physicalremoval of the etiological agent, the artisan will recognize that theyare equally effective in situations where the inventive compound isadministered prior to, or simultaneous with, exposure to the etiologicalagent (prophylactic treatment) and situations where the inventivecompounds are administered after (even well after) exposure to theetiological agent.

Treatment of cancer, as used herein, refers to partially or totallyinhibiting, delaying or preventing the progression of cancer includingcancer metastasis; inhibiting, delaying or preventing the recurrence ofcancer including cancer metastasis; or preventing the onset ordevelopment of cancer (chemoprevention) in a mammal, for example ahuman.

As used herein, the term “therapeutically effective amount” is intendedto encompass any amount that will achieve the desired therapeutic orbiological effect. The therapeutic effect is dependent upon the diseaseor disorder being treated or the biological effect desired. As such, thetherapeutic effect can be a decrease in the severity of symptomsassociated with the disease or disorder and/or inhibition (partial orcomplete) of progression of the disease. The amount needed to elicit thetherapeutic response can be determined based on the age, health, sizeand sex of the subject. Optimal amounts can also be determined based onmonitoring of the subject's response to treatment.

In the present invention, when the compounds are used to treat orprevent cancer, the desired biological response is partial or totalinhibition, delay or prevention of the progression of cancer includingcancer metastasis; inhibition, delay or prevention of the recurrence ofcancer including cancer metastasis; or the prevention of the onset ordevelopment of cancer (chemoprevention) in a mammal, for example ahuman.

Furthermore, in the present invention, when the compounds are used totreat and/or prevent thioredoxin (TRX)-mediated diseases and conditions,a therapeutically effective amount is an amount that regulates, forexample, increases, decreases or maintains a physiologically suitablelevel of TRX in the subject in need of treatment to elicit the desiredtherapeutic effect. The therapeutic effect is dependent upon thespecific TRX-mediated disease or condition being treated. As such, thetherapeutic effect can be a decrease in the severity of symptomsassociated with the disease or disorder and/or inhibition (partial orcomplete) of progression of the disease or disease.

Furthermore, in the present invention, when the compounds are used totreat and/or prevent diseases or disorders of the central nervous system(CNS), a therapeutically effective amount is dependent upon the specificdisease or disorder being treated. As such, the therapeutic effect canbe a decrease in the severity of symptoms associated with the disease ordisorder and/or inhibition (partial or complete) of progression of thedisease or disorder.

In addition, a therapeutically effective amount can be an amount thatinhibits histone deacetylase.

Further, a therapeutically effective amount, can be an amount thatselectively induces terminal differentiation, cell growth arrest and/orapoptosis of neoplastic cells, or an amount that induces terminaldifferentiation of tumor cells.

The method of the instant invention is intended for the treatment orchemoprevention of human patients with cancer. However, it is alsolikely that the method would be effective in the treatment of cancer inother subjects. “Subject”, as used herein, refers to animals such asmammals, including, but not limited to, primates (e.g., humans), cows,sheep, goats, horses, pigs, dogs, cats, rabbits, guinea pigs, rats, miceor other bovine, ovine, equine, canine, feline, rodent or murinespecies.

As demonstrated herein, the hydroxamic acid derivatives of the instantinvention show improved activity as histone deacetylase (HDAC)inhibitors. In one embodiment, the concentration of compound requiredfor 50% inhibition (IC₅₀) of histone deacetylase is below 1000 nM. Inanother embodiment, the concentration of compound required for 50%inhibition (IC₅₀) of histone deacetylase is between 500 and 1000 nM. Inanother embodiment, the concentration of compound required for 50%inhibition (IC₅₀) of histone deacetylase is between 100 and 500 mM. Inanother embodiment, the concentration of compound required for 50%inhibition (IC₅₀) of histone deacetylase is below 100 nM. In anotherembodiment, the concentration of compound required for 50% inhibition(IC₅₀) of histone deacetylase is between 10 and 100 nM. In anotherembodiment, the concentration of compound required for 50% inhibition(IC₅₀) of histone deacetylase is below 50 nM. In another embodiment, theconcentration of compound required for 50% inhibition (IC₅₀) of histonedeacetylase is between 10 and 50 nM. In another embodiment, theconcentration of compound required for 50% inhibition (IC₅₀) of histonedeacetylase is below 10 nM. In another embodiment, the concentration ofcompound required for 50% inhibition (IC₅₀) of histone deacetylase isbetween 1 and 10 nM. In another embodiment, the concentration ofcompound required for 50% inhibition (IC₅₀) of histone deacetylase isbelow 1 nM. In another embodiment, the concentration of compoundrequired for 50% inhibition (IC₅₀) of histone deacetylase is between 0.1and 1 nM.

Accordingly, in one embodiment, the invention relates to a method ofinhibiting the activity of histone deacetylase comprising contacting thehistone deacetylase with an effective amount of one or more of thehydroxamic acid compounds described herein.

In one embodiment, the hydroxamic acid derivatives are potent inhibitorsof Class I histone deacetylases (Class I HDACs). Class I HDACs includehistone deacetylase 1 (HDAC-1), histone deacetylase 2 (HDAC-2), histonedeacetylase 3 (HDAC-3) and histone deacetylase 8 (HDAC-8). In aparticular embodiment, the hydroxamic acid derivatives are potentinhibitors of histone deacetylase I (HDAC-1). In another embodiment, thehydroxamic acid derivatives are potent inhibitors of Class I histonedeacetylases (Class II HDACs). Class II HDACs include histonedeacetylase 4 (HDAC-4), histone deacetylase 5 (HDAC-8), histonedeacetylase 6 (HDAC-6), histone deacetylase 7 (HDAC-7) and histonedeacetylase 9 (HDAC-9).

Histone deacetylases (HDACs), as that term is used herein, are enzymesthat catalyze the removal of acetyl groups from lysine residues in theamino terminal tails of the nucleosomal core histones. As such, HDACstogether with histone acetyl transferases (HATs) regulate theacetylation status of histones. Histone acetylation affects geneexpression and inhibitors of HDACs, such as the hydroxamic acid-basedhybrid polar compound suberoylanilide hydroxamic acid (SAHA) inducegrowth arrest, differentiation and/or apoptosis of transformed cells invitro and inhibit tumor growth in vivo. HDACs can be divided into threeclasses based on structural homology. Class I HDACs (HDACs 1, 2, 3 and8) bear similarity to the yeast RPD3 protein, are located in the nucleusand are found in complexes associated with transcriptionalco-repressors. Class II HDACs (HDACs 4, 5, 6, 7 and 9) are similar tothe yeast HDA1 protein, and have both nuclear and cytoplasmicsubcellular localization. Both Class I and II HDACs are inhibited byhydroxamic acid-based HDAC inhibitors, such as SAHA. Class III HDACsform a structurally distant class of NAD dependent enzymes that arerelated to the yeast SIR2 proteins and are not inhibited by hydroxamicacid-based HDAC inhibitors.

Histone deacetylase inhibitors or HDAC inhibitors, as that term is usedherein are compounds that are capable of inhibiting the deacetylation ofhistones in vivo, in vitro or both. As such, HDAC inhibitors inhibit theactivity of at least one histone deacetylase. As a result of inhibitingthe deacetylation of at least one histone, an increase in acetylatedhistone occurs and accumulation of acetylated histone is a suitablebiological marker for assessing the activity of HDAC inhibitors.Therefore, procedures that can assay for the accumulation of acetylatedhistones can be used to determine the HDAC inhibitory activity ofcompounds of interest. It is understood that compounds that can inhibithistone deacetylase activity can also bind to other substrates and assuch can inhibit other biologically active molecules such as enzymes. Itis also to be understood that the compounds of the instant invention arecapable of inhibiting any of the histone deacetylases set forth above,or any other histone deacetylases.

For example, in patients receiving HDAC inhibitors, the accumulation ofacetylated histones in peripheral mononuclear cells as well as in tissuetreated with HDAC inhibitors can be determined against a suitablecontrol.

HDAC inhibitory activity of a particular compound can be determined invitro using, for example, an enzymatic assay which shows inhibition ofat least one histone deacetylase. Further, determination of theaccumulation of acetylated histones in cells treated with a particularcomposition can be determinative of the HDAC inhibitory activity of acompound.

Assays for the accumulation of acetylated histones are well known in theliterature. See, for example, Marks, P. A. et al., J. Natl. CancerInst., 92:1210-1215, 2000, Butler, L. M. et al., Cancer Res.60:5165-5170 (2000), Richon, V. M. et al., Proc. Natl. Acad. Sci., USA,95:3003-3007, 1998, and Yoshida, M. et al., J. Biol. Chem.,265:17174-17179, 1990.

For example, an enzymatic assay to determine the activity of an HDACinhibitor compound can be conducted as follows. Briefly, the effect ofan HDAC inhibitor compound on affinity purified human epitope-tagged(Flag) HDAC1 can be assayed by incubating the enzyme preparation in theabsence of substrate on ice for about 20 minutes with the indicatedamount of inhibitor compound. Substrate ([³H]acetyl-labelled murineerythroleukemia cell-derived histone) can be added and the sample can beincubated for 20 minutes at 37° C. in a total volume of 30 μL. Thereaction can then be stopped and released acetate can be extracted andthe amount of radioactivity release determined by scintillationcounting. An alternative assay useful for determining the activity of anHDAC inhibitor compound is the “HDAC Fluorescent Activity Assay; DrugDiscovery Kit-AK-500” available from BIOMOL® Research Laboratories,Inc., Plymouth Meeting, Pa.

In vivo studies can be conducted as follows. Animals, for example, mice,can be injected intraperitoneally with an HDAC inhibitor compound.Selected tissues, for example, brain, spleen, liver etc, can be isolatedat predetermined times, post administration. Histones can be isolatedfrom tissues essentially as described by Yoshida et al., J. Biol. Chem.265:17174-17179, 1990. Equal amounts of histones (about 1 μg) can beelectrophoresed on 15% SDS-polyacrylamide gels and can be transferred toHybond-P filters (available from Amersham). Filters can be blocked with3% milk and can be probed with a rabbit purified polyclonalanti-acetylated histone H4 antibody (αAc-H4) and anti-acetylated histoneH3 antibody (αAc-H3) (Upstate Biotechnology, Inc.). Levels of acetylatedhistone can be visualized using a horseradish peroxidase-conjugated goatanti-rabbit antibody (1:5000) and the SuperSignal chemiluminescentsubstrate (Pierce). As a loading control for the histone protein,parallel gels can be run and stained with Coomassie Blue (CB).

In addition, hydroxamic acid-based HDAC inhibitors have been shown to upregulate the expression of the p21^(WAF1) gene. The p21^(WAF1) proteinis induced within 2 hours of culture with HDAC inhibitors in a varietyof transformed cells using standard methods. The induction of thep21^(WAF1) gene is associated with accumulation of acetylated histonesin the chromatin region of this gene. Induction of p21^(WAF1) cantherefore be recognized as involved in the G1 cell cycle arrest causedby HDAC inhibitors in transformed cells.

Typically, HDAC inhibitors fall into five general classes: 1) hydroxamicacid derivatives; 2) Short-Chain Fatty Acids (SCFAs); 3) cyclictetrapeptides; 4) benzamides; and 5) electrophilic ketones. Examples ofsuch HDAC inhibitors are set forth below.

A. Hydroxamic Acid Derivatives such as: suberoylanilide hydroxamic acid(SAHA) (Richon et al., Proc. Natl. Acad. Sci. USA 95, 3003-3007 (1998));m-carboxycinnamic acid bishydroxamide (CBHA) (Richon et al., supra);pyroxamide; trichostatin analogues such as trichostatin A (TSA) andtrichostatin C (Koghe et al. 1998. Biochem. Pharmacol. 56: 1359-1364);salicylhydroxamic acid (Andrews et al., International J. Parasitology30, 761-768 (2000)); suberoyl bishydroxamic acid (SBHA) (U.S. Pat. No.5,608,108); azelaic bishydroxamic acid (ABHA) (Andrews et al., supra);azelaic-1-hydroxamate-9-anilide (AAHA) (Qiu et al., Mol. Biol. Cell 11,2069-2083 (2000)); 6-(3-chlorophenylureido) carpoic hydroxamic acid(3Cl-UCHA); oxamflatin[(2E)-5-[3-[(phenylsulfonyl)amino]phenyl]-pent-2-en-4-ynohydroxamicacid] (Kim et al. Oncogene, 18: 2461 2470 (1999)); A-161906, Scriptaid(Su et al. 2000 Cancer Research, 60: 3137-3142); PXD-101 (Prolifix);LAQ-824; CHAP; MW2796 (Andrews et al., supra); MW2996 (Andrews et al.,supra); or any of the hydroxamic acids disclosed in U.S. Pat. Nos.5,369,108, 5,932,616, 5,700,811, 6,087,367 and 6,511,990.B. Cyclic Tetrapeptides such as: trapoxin A (TPX)-cyclic tetrapeptide(cyclo-(L-phenylalanyl-L-phenylalanyl-D-pipecolinyl-L-2-amino-8-oxo-9,10-epoxydecanoyl)) (Kijima et al., J. Biol. Chem. 268, 22429-22435 (1993));FR901228 (FK 228, depsipeptide) (Nakajima et al., Ex. Cell Res. 241,126-133 (1998)); FR225497 cyclic tetrapeptide (H. Mori et al., PCTApplication WO 00/08048 (17 Feb. 2000)); apicidin cyclic tetrapeptide[cyclo(N—O-methyl-L-tryptophanyl-L-isoleucinyl-D-pipecolinyl-L-2-amino-8-oxodecanoyl)](Darkin-Rattray et al., Proc. Natl. Acad. Sci. USA 93, 1314313147(1996)); apicidin Ia, apicidin Ib, apicidin Ic, apicidin IIa, andapicidin IIb (P. Dulski et al., PCT Application WO 97/11366); CHAP,HC-toxin cyclic tetrapeptide (Bosch et al., Plant Cell 7, 1941-1950(1995)); WF27082 cyclic tetrapeptide (PCT Application WO 98/48825); andchlamydocin (Bosch et al., supra).C. Short chain fatty acid (SCFA) derivatives such as: sodium butyrate(Cousens et al., J. Biol. Chem. 254, 1716-1723 (1979)); isovalerate(McBain et al., Biochem. Pharm. 53: 1357-1368 (1997)); valerate (McBainet al., supra); 4-phenylbutyrate (4-PBA) (Lea and Tulsyan, AnticancerResearch, 15, 879-873 (1995)); phenylbutyrate (PB) (Wang et al., CancerResearch, 59, 2766-2799 (1999)); propionate (McBain et al., supra);butyramide (Lea and Tulsyan, supra); isobutyramide (Lea and Tulsyan,supra); phenylacetate (Lea and Tulsyan, supra); 3-bromopropionate (Leaand Tulsyan, supra); tributyrin (Guan et al., Cancer Research, 60,749-755 (2000)); valproic acid, valproate and Pivanex™.D. Benzamide derivatives such as: CI-994; MS-275[N-(2-aminophenyl)-4-[N-(pyridin-3-ylmethoxycarbonyl)aminomethyl]benzamide](Saito et al., Proc. Natl. Acad. Sci. USA 96, 4592-4597 (1999)); and3′-amino derivative of MS-275 (Saito et al., supra).E. Electrophilic ketone derivatives such as: trifluoromethyl ketones(Frey et al, Bioorganic & Med. Chem. Lett. (2002), 12, 3443-3447; U.S.Pat. No. 6,511,990) and α-keto amides such as N-methyl-α-ketoamidesF. Other HDAC Inhibitors such as: natural products, psammaplins andDepudecin (Kwon et al. 1998. PNAS 95: 3356-3361).

The hydroxamic acid compounds of the instant invention can beadministered alone or in combination with other therapies suitable forthe disease or disorder being treated. Where separate dosageformulations are used, the hydroxamic acid compound and the othertherapeutic agent can be administered at essentially the same time(concurrently) or at separately staggered times (sequentially). Thepharmaceutical combination is understood to include all these regimens.Administration in these various ways are suitable for the presentinvention as long as the beneficial therapeutic effect of the hydroxamicacid compound and the other therapeutic agent are realized by thepatient at substantially the same time. Such beneficial effect ispreferably achieved when the target blood level concentrations of eachactive drug are maintained at substantially the same time.

A person of skill in the art would be able to discern which combinationsof agents would be useful based on the disease being treated, e.g. whichcancer, which neurodegenerative disease or which inflammatory disease isinvolved.

The hydroxamic acid derivatives can be administered in combination withany one or more of an HDAC inhibitor (e.g. any one or more of the HDACinhibitors described above), an alkylating agent, an antibiotic agent,an antimetabolic agent, a hormonal agent, a plant-derived agent, abiologic agent, a gene therapy agent, an anti-angiogenic agent, adifferentiation inducing agent, a retinoid receptor modulator, acytotoxic/cytostatic agent, an anti-proliferative agent, an HMG-CoAreductase inhibitor, a prenyl-protein transferase inhibitor, an agentthat interferes with cell cycle checkpoints, an inhibitor of cellproliferation and survival signaling, an apoptosis inducing agent, acell growth arrest inducing agent, or any combination thereof. Inaddition, the instant compounds are particularly useful whenco-administered with radiation therapy.

“Alkylating agents” react with nucleophilic residues, such as thechemical entities on the nucleotide precursors for DNA production. Theyaffect the process of cell division by alkylating these nucleotides andpreventing their assembly into DNA.

Examples of alkylating agents include, but are not limited to,bischloroethylamines (nitrogen mustards, e.g., chlorambucil,cyclophosphamide, ifosfamide, mechlorethamine, melphalan, uracilmustard), aziridines (e.g., thiotepa), alkyl alkone sulfonates (e.g.,busulfan), nitrosoureas (e.g., carmustine, lomustine, streptozocin),nonclassic alkylating agents (altretamine, dacarbazine, andprocarbazine), platinum compounds (carboplatin and cisplatin). Thesecompounds react with phosphate, amino, hydroxyl, sulfihydryl, carboxyl,and imidazole groups.

Under physiological conditions, these drugs ionize and producepositively charged ion that attach to susceptible nucleic acids andproteins, leading to cell cycle arrest and/or cell death. The alkylatingagents are cell cycle phase nonspecific agents because they exert theiractivity independently of the specific phase of the cell cycle. Thenitrogen mustards and alkyl alkone sulfonates are most effective againstcells in the G₁ or M phase. Nitrosoureas, nitrogen mustards, andaziridines impair progression from the G₁ and S phases to the M phases.Chabner and Collins eds. (1990) “Cancer Chemotherapy: Principles andPractice”, Philadelphia: J B Lippincott.

The alkylating agents are active against wide variety of neoplasticdiseases, with significant activity in the treatment of leukemias andlymphomas as well as solid tumors. Clinically this group of drugs isroutinely used in the treatment of acute and chronic leukemias;Hodgkin's disease; non-Hodgkin's lymphoma; multiple myeloma; primarybrain tumors; carcinomas of the breast, ovaries, testes, lungs, bladder,cervix, head and neck, and malignant melanoma.

“Antibiotic agents” (e.g., cytotoxic antibiotics) act by directlyinhibiting DNA or RNA synthesis and are effective throughout the cellcycle. Examples of antibiotic agents include anthracyclines (e.g.,doxorubicin, daunorubicin, epirubicin, idarubicin and anthracenedione),mitomycin C, bleomycin, dactinomycin, and plicatomycin. These antibioticagents interfere with cell growth by targeting different cellularcomponents. For example, anthracyclines are generally believed tointerfere with the action of DNA topoisomerase II in the regions oftranscriptionally active DNA, which leads to DNA strand scissions.

Bleomycin is generally believed to chelate iron and forms an activatedcomplex, which then binds to bases of DNA, causing strand scissions andcell death.

The antibiotic agents have been used as therapeutics across a range ofneoplastic diseases, including carcinomas of the breast, lung, stomachand thyroids, lymphomas, myelogenous leukemias, myelomas, and sarcomas.

“Antimetabolic agents” (i.e., antimetabolites) are a group of drugs thatinterfere with metabolic processes vital to the physiology andproliferation of cancer cells. Actively proliferating cancer cellsrequire continuous synthesis of large quantities of nucleic acids,proteins, lipids, and other vital cellular constituents.

Many of the antimetabolites inhibit the synthesis of purine orpyrimidine nucleosides or inhibit the enzymes of DNA replication. Someantimetabolites also interfere with the synthesis of ribonucleosides andRNA and/or amino acid metabolism and protein synthesis as well. Byinterfering with the synthesis of vital cellular constituents,antimetabolites can delay or arrest the growth of cancer cells. Examplesof antimetabolic agents include, but are not limited to, fluorouracil(5-FU), floxuridine (5-FUdR), methotrexate, leucovorin, hydroxyurea,thioguanine (6-TG), mercaptopurine (6-MP), cytarabine, pentostatin,fludarabine phosphate, cladribine (2-CDA), asparaginase, andgemcitabine.

Antimetabolic agents have widely used to treat several common forms ofcancer including carcinomas of colon, rectum, breast, liver, stomach andpancreas, malignant melanoma, acute and chronic leukemia and hair cellleukemia.

“Hormonal agents” are a group of drugs that regulate the growth anddevelopment of their target organs. Some of the hormonal agents are sexsteroids and their derivatives and analogs thereof, such as estrogens,progestogens, anti-estrogens, androgens, anti-androgens and progestins.Other hormonal agents are small molecules that regulate their targetreceptors. These hormonal agents may serve as antagonists of receptorsfor the sex steroids to down regulate receptor expression andtranscription of vital genes. Examples of such hormonal agents aresynthetic estrogens (e.g., diethylstibestrol), estrogen receptormodulators, selective estrogen receptor modulators (SERMs),antiestrogens (e.g., tamoxifen, toremifene, fluoxymesterol andraloxifene), androgen receptor modulators, selective androgen receptormodulators (SARM), antiandrogens (e.g. bicalutamide, nilutamide,flutamide), aromatase inhibitors (e.g., aminoglutethimide, anastrozoleand tetrazole), luteinizing hormone release hormone (LHRH) analogues,ketoconazole, goserelin acetate, leuprolide, megestrol acetate andmifepristone.

Hormonal agents are used to treat breast cancer, prostate cancer,melanoma and meningioma. Because the major action of hormones ismediated through steroid receptors, 60% receptor-positive breast cancerresponded to first-line hormonal therapy; and less than 10% ofreceptor-negative tumors responded. Specifically, progestogens are usedto treat endometrial cancers, since these cancers occur in women thatare exposed to high levels of oestrogen unopposed by progestogen.Antiandrogens are used primarily for the treatment of prostate cancer,which is hormone dependent. They are used to decrease levels oftestosterone, and thereby inhibit growth of the tumor.

Hormonal treatment of breast cancer involves reducing the level ofoestrogen-dependent activation of oestrogen receptors in neoplasticbreast cells. Anti-oestrogens act by binding to oestrogen receptors andprevent the recruitment of coactivators, thus inhibiting the oestrogensignal.

LHRH analogues are used in the treatment of prostate cancer to decreaselevels of testosterone and so decrease the growth of the tumor.

Aromatase inhibitors act by inhibiting the enzyme required for hormonesynthesis. In post-menopausal women, the main source of oestrogen isthrough the conversion of androstenedione by aromatase.

Estrogen receptor modulators refers to compounds that interfere with orinhibit the binding of estrogen to the receptor, regardless ofmechanism. Examples of estrogen receptor modulators include, but are notlimited to, tamoxifen, raloxifene, idoxifene, LY353381, LY117081,toremifene, fulvestrant,4-[7-(2,2-dimethyl-1-oxopropoxy-4-methyl-2-[4-[2-(1-piperidinyl)ethoxy]phenyl]-2H-1-benzopyran-3-yl]-phenyl-2,2-dimethylpropanoate,4,4′-dihydroxybenzophenone-2,4-dinitrophenyl-hydrazone, and SH646.

Androgen receptor modulators refers to compounds which interfere orinhibit the binding of androgens to the receptor, regardless ofmechanism. Examples of androgen receptor modulators include finasterideand other 5α-reductase inhibitors, nilutamide, flutamide, bicalutamide,liarozole, and abiraterone acetate.

“Plant-derived agents” are a group of drugs that are derived from plantsor modified based on the molecular structure of the agents. They inhibitcell replication by preventing the assembly of the cell's componentsthat are essential to cell division.

Examples of plant derived agents include vinca alkaloids (e.g.,vincristine, vinblastine, vindesine, vinzolidine and vinorelbine),podophyllotoxins (e.g., etoposide (VP-16) and teniposide (VM-26)),taxanes (e.g., paclitaxel and docetaxel). These plant-derived agentsgenerally act as antimitotic agents that bind to tubulin and inhibitmitosis. Podophyllotoxins such as etoposide are believed to interferewith DNA synthesis by interacting with topoisomerase II, leading to DNAstrand scission.

Plant-derived agents are used to treat many forms of cancer. Forexample, vincristine is used in the treatment of the leukemias,Hodgkin's and non-Hodgkin's lymphoma, and the childhood tumorsneuroblastoma, rhabdomyosarcoma, and Wilm's tumor. Vinblastine is usedagainst the lymphomas, testicular cancer, renal cell carcinoma, mycosisfungoides, and Kaposi's sarcoma. Doxetaxel has shown promising activityagainst advanced breast cancer, non-small cell lung cancer (NSCLC), andovarian cancer.

Etoposide is active against a wide range of neoplasms, of which smallcell lung cancer, testicular cancer, and NSCLC are most responsive.

“Biologic agents” are a group of biomolecules that elicit cancer/tumorregression when used alone or in combination with chemotherapy and/orradiotherapy. Examples of biologic agents include immuno-modulatingproteins such as cytokines, monoclonal antibodies against tumorantigens, tumor suppressor genes, and cancer vaccines.

Cytokines possess profound immunomodulatory activity. Some cytokinessuch as interleukin-2 (IL-2, aldesleukin) and interferon-α (IFN-α)demonstrated antitumor activity and have been approved for the treatmentof patients with metastatic renal cell carcinoma and metastaticmalignant melanoma. IL-2 is a T-cell growth factor that is central toT-cell-mediated immune responses. The selective antitumor effects ofEL-2 on some patients are believed to be the result of a cell-mediatedimmune response that discriminate between self and nonself.

Interferon-α includes more than 23 related subtypes with overlappingactivities. IFN-a has demonstrated activity against many solid andhematologic malignancies, the later appearing to be particularlysensitive.

Examples of interferons include, interferon-α, interferon-β (fibroblastinterferon) and interferon-γ (fibroblast interferon). Examples of othercytokines include erythropoietin (epoietin-α), granulocyte-CSF(filgrastin), and granulocyte, macrophage-CSF (sargramostim). Otherimmuno-modulating agents other than cytokines include bacillusCalmette-Guerin, levamisole, and octreotide, a long-acting octapeptidethat mimics the effects of the naturally occurring hormone somatostatin.

Furthermore, the anti-cancer treatment can comprise treatment byimmunotherapy with antibodies and reagents used in tumor vaccinationapproaches. The primary drugs in this therapy class are antibodies,alone or carrying compounds such as toxins orchemotherapeutics/cytotoxics to cancer cells. Monoclonal antibodiesagainst tumor antigens are antibodies elicited against antigensexpressed by tumors, preferably tumor-specific antigens. For example,monoclonal antibody HERCEPTIN® (trastuzumab) is raised against humanepidermal growth factor receptor2 (HER2) that is overexpressed in somebreast tumors including metastatic breast cancer. Overexpression of HER2protein is associated with more aggressive disease and poorer prognosisin the clinic. HERCEPTIN® is used as a single agent for the treatment ofpatients with metastatic breast cancer whose tumors over express theHER2 protein.

Another example of monoclonal antibodies against tumor antigens isRITUXAN® (rituximab) that is raised against CD20 on lymphoma cells andselectively deplete normal and malignant CD20+ pre-B and mature B cells.

RITUXAN is used as single agent for the treatment of patients withrelapsed or refractory low-grade or follicular, CD20+, B cellnon-Hodgkin's lymphoma. MYELOTARG® (gemtuzumab ozogamicin) and CAMPATH®(alemtuzumab) are further examples of monoclonal antibodies againsttumor antigens that may be used.

In addition, examples of monoclonal antibody targeted therapeutic agentsinclude those therapeutic agents which have cytotoxic agents orradioisotopes attached to a cancer cell specific or target cell specificmonoclonal antibody. Examples include Bexxar.

Tumor suppressor genes are genes that function to inhibit the cellgrowth and division cycles, thus preventing the development ofneoplasia. Mutations in tumor suppressor genes cause the cell to ignoreone or more of the components of the network of inhibitory signals,overcoming the cell cycle checkpoints and resulting in a higher rate ofcontrolled cell growth-cancer. Examples of the tumor suppressor genesinclude Duc-4, NF-1, NF-2, RB, p53, WT1, BRCA1 and BRCA2.

DPC4 is involved in pancreatic cancer and participates in a cytoplasmicpathway that inhibits cell division. NF-1 codes for a protein thatinhibits Ras, a cytoplasmic inhibitory protein. NF-1 is involved inneurofibroma and pheochromocytomas of the nervous system and myeloidleukemia. NF-2 encodes a nuclear protein that is involved in meningioma,schwanoma, and ependymoma of the nervous system. RB codes for the pRBprotein, a nuclear protein that is a major inhibitor of cell cycle. RBis involved in retinoblastoma as well as bone, bladder, small cell lungand breast cancer. P53 codes for p53 protein that regulates celldivision and can induce apoptosis. Mutation and/or inaction of p53 isfound in a wide ranges of cancers. WTI is involved in Wilm's tumor ofthe kidneys. BRCA1 is involved in breast and ovarian cancer, and BRCA2is involved in breast cancer. The tumor suppressor gene can betransferred into the tumor cells where it exerts its tumor suppressingfunctions.

Cancer vaccines are a group of agents that induce the body's specificimmune response to tumors. Most of cancer vaccines under research anddevelopment and clinical trials are tumor-associated antigens (TAAs).TAAs are structures (i.e., proteins, enzymes or carbohydrates) that arepresent on tumor cells and relatively absent or diminished on normalcells. By virtue of being fairly unique to the tumor cell, TAAs providetargets for the immune system to recognize and cause their destruction.Examples of TAAs include gangliosides (GM2), prostate specific antigen(PSA), α-fetoprotein (AFP), carcinoembryonic antigen (CEA) (produced bycolon cancers and other adenocarcinomas, e.g., breast, lung, gastric,and pancreatic cancers), melanoma-associated antigens (MART-1, gap100,MAGE 1,3 tyrosinase), papillomavirus E6 and E7 fragments, whole cells orportions/lysates of autologous tumor cells and allogeneic tumor cells.

Recent developments have introduced, in addition to the traditionalcytotoxic and hormonal therapies used to treat cancer, additionaltherapies for the treatment of cancer. For example, many forms of genetherapy are undergoing preclinical or clinical trials. Thus, anotherembodiment of the instant invention is the use of the presentlydisclosed compounds in combination with gene therapy for the treatmentof cancer. For an overview of genetic strategies to treating cancer seeHall et al (Am J Hum Genet. 61:785-789, 1997) and Kufe et al (CancerMedicine, 5th Ed, pp 876-889, BC Decker, Hamilton 2000). Gene therapycan be used to deliver any tumor suppressing gene. Examples of suchgenes include, but are not limited to, p53, which can be delivered viarecombinant virus-mediated gene transfer (see U.S. Pat. No. 6,069,134,for example), a uPA/uPAR antagonist (“Adenovirus-Mediated Delivery of auPA/uPAR Antagonist Suppresses Angiogenesis-Dependent Tumor Growth andDissemination in Mice,” Gene Therapy, August 1998; 5(8):1105-13), andinterferon gamma (J Immunol 2000; 164:217-222).

In addition, approaches are currently under development, that are basedon the inhibition of tumor vascularization (angiogenesis). The aim ofthis concept is to cut off the tumor from nutrition and oxygen supplyprovided by a newly built tumor vascular system. “Angiogenesisinhibitors” refers to compounds that inhibit the formation of new bloodvessels, regardless of mechanism. Examples of angiogenesis inhibitorsinclude, but are not limited to, tyrosine kinase inhibitors, such asinhibitors of the tyrosine kinase receptors Flt-1 (VEGFR1) and Flk-1/KDR(VEGFR2), inhibitors of epidermal-derived, fibroblast-derived, orplatelet derived growth factors, MMP (matrix metalloprotease)inhibitors, integrin blockers, interferon-α, interleukin-12, pentosanpolysulfate, cyclooxygenase inhibitors, including nonsteroidalanti-inflammatories (NSAIDs) like aspirin and ibuprofen as well asselective cyclooxy-genase-2 inhibitors like celecoxib and rofecoxib(PNAS, Vol. 89, p. 7384 (1992); JNCI, Vol. 69, p. 475 (1982); Arch.Opthalmol., Vol. 108, p. 573 (1990); Anat. Rec., Vol. 238, p. 68 (1994);FEBS Letters, Vol. 372, p. 83 (1995); Clin, Orthop. Vol. 313, p. 76(1995); J. Mol. Endocrinol., Vol. 16, p. 107 (1996); Jpn. J. Pharmacol.,Vol. 75, p. 105 (1997); Cancer Res., Vol. 57, p. 1625 (1997); Cell, Vol.93, p. 705 (1998); Intl. J. Mol. Med., Vol. 2, p. 715 (1998); J. Biol.Chem., Vol. 274, p. 9116 (1999)), steroidal anti-inflammatories (such ascorticosteroids, mineralocorticoids, dexamethasone, prednisone,prednisolone, methylpred, betamethasone), carboxyamidotriazole,combretastatin A-4, squalamine, 6-O-chloroacetyl-carbonyl)-fumagillol,thalidomide, angiostatin, troponin-1, angiotensin II antagonists (seeFernandez et al., J. Lab. Clin. Med. 105:141-145 (1985)), and antibodiesto VEGF (see, Nature Biotechnology, Vol. 17, pp. 963-968 (October 1999);Kim et al., Nature, 362, 841-844 (1993); WO 00/44777; and WO 00/61186).

Other therapeutic agents that modulate or inhibit angiogenesis and mayalso be used in combination with the compounds of the instant inventioninclude agents that modulate or inhibit the coagulation and fibrinolysissystems (see review in Clin. Chem. La. Med. 38:679-692 (2000)). Examplesof such agents that modulate or inhibit the coagulation and fibrinolysispathways include, but are not limited to, heparin (see Thromb. Haemost.80:10-23 (1998)), low molecular weight heparins and carboxypeptidase Uinhibitors (also known as inhibitors of active thrombin activatablefibrinolysis inhibitor [TAFIa]) (see Thrombosis Res. 101:329-354(2001)). TAFIa inhibitors have been described in PCT Publication WO03/013,526 and U.S. Ser. No. 60/349,925 (filed Jan. 18, 2002).

Other examples of angiogenesis inhibitors include, but are not limitedto, endostatin, ukrain, ranpirnase, IM862,5-methoxy-4-[2-methyl-3-(3-methyl-2-butenyl)oxiranyl]-1-oxaspiro[2,5]oct-6-yl(chloroacetyl)carbamate,acetyldinanaline,5-amino-1-[[3,5-dichloro-4-(4-chlorobenzoyl)phenyl]methyl]-1H-1,2,3-triazole-4-carboxamide,CM101, squalamine, combretastatin, RPI4610, NX31838, sulfatedmannopentaose phosphate,7,7-(carbonyl-bis[imino-N-methyl-4,2-pyrrolocarbonylimino[N-methyl-4,2-pyrrole]-carbonylimino]-bis-(1,3-naphthalenedisulfonate), and 3-[(2,4-dimethylpyrrol-5-yl)methylene]-2-indolinone(SU5416).

In addition, cancer therapy is also being attempted by the induction ofterminal differentiation of the neoplastic cells. Suitabledifferentiation agents include the compounds disclosed in any one ormore of the following references, the contents of which are incorporatedby reference herein.

a) Polar compounds (Marks et al (1987); , Friend, C., Scher, W.,Holland, J. W., and Sato, T. (1971) Proc. Natl. Acad. Sci. (USA) 68:378-382; Tanaka, M., Levy, J., Terada, M., Breslow, R., Rilkind, R. A.,and Marks, P. A. (1975) Proc. Natl. Acad. Sci. (USA) 72: 1003-1006;Reuben, R. C., Wife, R. L., Breslow, R., Rifkind, R. A., and Marks, P.A. (1976) Proc. Natl. Acad. Sci. (USA) 73: 862-866);

b) Derivatives of vitamin D and retinoic acid (Abe, E., Miyaura, C.,Sakagami, H., Takeda, M., Konno, K., Yamazaki, T., Yoshika, S., andSuda, T. (1981) Proc. Natl. Acad. Sci. (USA) 78: 4990-4994; Schwartz, E.L., Snoddy, J. R., Kreutter, D., Rasmussen, H., and Sartorelli, A. C.(1983) Proc. Am. Assoc. Cancer Res. 24: 18; Tanenaga, K., Hozumi, M.,and Sakagami, Y. (1980) Cancer Res. 40: 914-919);

c) Steroid hormones (Lotem, J. and Sachs, L. (1975) Int. J. Cancer 15:731-740);

d) Growth factors (Sachs, L. (1978) Nature (Lond.) 274: 535, Metcalf, D.(1985) Science, 229: 16-22);

e) Proteases (Scher, W., Scher, B. M., and Waxman, S. (1983) Exp.Hematol. 11: 490-498; Scher, W., Scher, B. M., and Waxman, S. (1982)Biochem. & Biophys. Res. Comm. 109: 348-354);

f) Tumor promoters (Huberman, E. and Callaham, M. F. (1979) Proc. Natl.Acad. Sci. (USA) 76: 1293-1297; Lottem, J. and Sachs, L. (1979) Proc.Natl. Acad. Sci. (USA) 76: 5158-5162); and

g) inhibitors of DNA or RNA synthesis (Schwartz, E. L. and Sartorelli,A. C. (1982) Cancer Res. 42: 2651-2655, Terada, M., Epner, E., Nudel,U., Salmon, J., Fibach, E., Rifkind, R. A., and Marks, P. A. (1978)Proc. Natl. Acad. Sci. (USA) 75: 2795-2799; Morin, M. J. and Sartorelli,A. C. (1984) Cancer Res. 44: 2807-2812; Schwartz, E. L., Brown, B. J.,Nierenberg, M., Marsh, J. C., and Sartorelli, A. C. (1983) Cancer Res.43: 2725-2730; Sugano, H., Furusawa, M., Kawaguchi, T., and Ikawa, Y.(1973) Bibl. Hematol. 39: 943-954; Ebert, P. S., Wars, I., and Buell, D.N. (1976) Cancer Res. 36: 1809-1813; Hayashi, M., Okabe, J., and Hozumi,M. (1979) Gann 70: 235-238).

“Retinoid receptor modulators” refers to compounds which interfere orinhibit the binding of retinoids to the receptor, regardless ofmechanism. Examples of such retinoid receptor modulators includebexarotene, tretinoin, 13-cis-retinoic acid, 9-cis-retinoic acid,α-difluoromethylornithine, ILX23-7553, trans-N-(4′-hydroxyphenyl)retinamide, and N-4-carboxyphenyl retinamide.

“Cytotoxic/cytostatic agents” refer to compounds which cause cell deathor inhibit cell proliferation primarily by interfering directly with thecell's functioning or inhibit or interfere with cell mytosis, includingalkylating agents (such as those described above), tumor necrosisfactors, intercalators, hypoxia activatable compounds, microtubuleinhibitors/microtubule-stabilizing agents, inhibitors of mitotickinesins, inhibitors of kinases involved in mitotic progression,haematopoietic growth factors, topoisomerase inhibitors, biologicalresponse modifiers, proteasome inhibitors and ubiquitin ligaseinhibitors. In addition, cyclotoxic/cytostatic agents include any of theantimetabolites; hormonal/anti-hormonal therapeutic agents, andmonoclonal antibody targeted therapeutic agents described above.

Examples of cytotoxic agents include, but are not limited to, sertenef,cachectin, ifosfamide, tasonermin, lonidamine, altretamine,prednimustine, dibromodulcitol, ranimustine, fotemustine, nedaplatin,oxaliplatin, temozolomide, heptaplatin, estramustine, improsulfantosilate, trofosfamide, nimustine, dibrospidium chloride, pumitepa,lobaplatin, satraplatin, profiromycin, cisplatin, irofulven,dexifosfamide, cis-aminedichloro(2-methyl-pyridine)platinum,benzylguanine, glufosfamide, GPX100, (trans, trans,trans)-bis-mu-(hexane-1,6-diamine)-mu-[diamine-platinumII]bis[diamine(chloro)platinumH]tetrachloride, diarizidinylspermine, arsenic trioxide,1-(11-dodecylamino-10-hydroxyundecyl)-3,7-dimethylxanthine, zorubicin,idarubicin, daunorubicin, bisantrene, mitoxantrone, pirarubicin,pinafide, valrubicin, amrubicin, antineoplaston,3′-deamino-3′-morpholino-13-deoxo-10-hydroxycaminomycin, annamycin,galarubicin, elinafide, MEN10755, and4-demethoxy-3-deamino-3-aziridinyl-4-methylsulphonyl-daunorubicin (seeWO 00/50032).

An example of a hypoxia activatable compound is tirapazamine.

Examples of proteasome inhibitors include but are not limited tolactacystin and bortezomib.

Examples of microtubule inhibitors/microtubule-stabilising agentsinclude paclitaxel, vindesine sulfate,3′,4′-didehydro-4′-deoxy-8′-norvincaleukoblastine, docetaxol, rhizoxin,dolastatin, mivobulin isethionate, auristatin, cemadotin, RPR109881,BMS184476, vinflunine, cryptophycin,2,3,4,5,6-pentafluoro-N-(3-fluoro-4-methoxyphenyl)benzene sulfonamide,anhydrovinblastine,N,N-dimethyl-L-valyl-L-valyl-N-methyl-L-valyl-L-prolyl-L-proline-t-butylamide,TDX258, the epothilones (see for example U.S. Pat. Nos. 6,284,781 and6,288,237) and BMS188797.

Some examples of topoisomerase inhibitors are topotecan, hycaptamine,irinotecan, rubitecan,6-ethoxypropionyl-3′,4′-O-exo-benzylidene-chartreusin,9-methoxy-N,N-dimethyl-5-nitropyrazolo[3,4,5-kl]acridine-2-(6H)propanamine,1-amino-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4-methyl-1H,12H-benzo[de]pyrano[3′,4′:b,7]-indolizino[1,2b]quinoline-10,13(9H,15H)dione,lurtotecan, 7-[2-(N-isopropylamino)ethyl]-(20S)camptothecin, BNP1350,BNPI1100, BN80915, BN80942, etoposide phosphate, teniposide, sobuzoxane,2′-dimethylamino-2′-deoxy-etoposide, GL331,N-[2-(dimethylamino)ethyl]-9-hydroxy-5,6-dimethyl-6H-pyrido[4,3-b]carbazole-1-carboxamide,asulacrine, (5a, 5aB,8aa,9b)-9-[2-[N-[2-(dimethylamino)ethyl]-N-methylamino]ethyl]-5-[4-hydro0xy-3,5-dimethoxyphenyl]-5,5a,6,8,8a,9-hexohydrofuro(3′,4′:6,7)naphtho(2,3-d)-1,3-dioxol-6-one,2,3-(methylenedioxy)-5-methyl-7-hydroxy-8-methoxybenzo[c]-phenanthridinium,6,9-bis[(2-aminoethyl)amino]benzo[g]isoquinoline-5,10-dione,5-(3-aminopropylamino)-7,10-dihydroxy-2-(2-hydroxyethylaminomethyl)-6H-pyrazolo[4,5,1-de]acridin-6-one,N-[1-[2(diethylamino)ethylamino]-7-methoxy-9-oxo-9H-thioxanthen-4-ylmethyl]formamide,N-(2-(dimethylamino)ethyl)acridine-4-carboxamide,6-[[2-(dimethylamino)ethyl]amino]-3-hydroxy-7H-indeno[2,1-c]quinolin-7-one,and dimesna.

Examples of inhibitors of mitotic kinesins, and in particular the humanmitotic kinesin KSP, are described in PCT Publications WO 01/30768, WO01/98278, WO 03/050,064, WO 03/050,122, WO 03/049,527, WO 03/049,679, WO03/049,678 and WO 03/39460 and pending PCT Appl. Nos. US03/06403 (filedMar. 4, 2003), US03/15861 (filed May 19, 2003), US03/15810 (filed May19, 2003), US03/18482 (filed Jun. 12, 2003) and US03/18694 (filed Jun.12, 2003). In an embodiment inhibitors of mitotic kinesins include, butare not limited to inhibitors of KSP, inhibitors of MKLP1, inhibitors ofCENP-E, inhibitors of MCAK, inhibitors of Kif14, inhibitors of Mphosph1and inhibitors of Rab6-KIFL.

“Inhibitors of kinases involved in mitotic progression” include, but arenot limited to, inhibitors of aurora kinase, inhibitors of Polo-likekinases (PLK) (in particular inhibitors of PLK-1), inhibitors of bub-1and inhibitors of bub-R1.

“Antiproliferative agents” includes antisense RNA and DNAoligonucleotides such as G3139, ODN698, RVASKRAS, GEM231, and INX3001,and antimetabolites such as enocitabine, carmofur, tegafur, pentostatin,doxifluridine, trimetrexate, fludarabine, capecitabine, galocitabine,cytarabine ocfosfate, fosteabine sodium hydrate, raltitrexed,paltitrexid, emitefur, tiazofurin, decitabine, nolatrexed, pemetrexed,nelzarabine, 2′-deoxy-2′-methylidenecytidine,2′-fluoromethylene-2′-deoxycytidine,N-[5-(2,3-dihydro-benzofuryl)sulfonyl]-N′-(3,4-dichlorophenyl)urea,N⁶-[4-deoxy-4-[N2-[2(E),4(E)-tetradecadienoyl]glycylamino]-L-glycero-B-L-manno-heptopyranosyl]adenine,aplidine, ecteinascidin, troxacitabine,4-[2-amino-4-oxo-4,6,7,8-tetrahydro-3H-pyrimidino[5,4-b][1,4]thiazin-6-yl-(S)-ethyl]-2,5-thienoyl-L-glutamicacid, aminopterin, 5-fluorouracil, alanosine,11-acetyl-8-(carbamoyloxymethyl)-4-formyl-6-methoxy-14-oxa-1,1′-diazatetracyclo(7.4.1.0.0)-tetradeca-2,4,6-trien-9-ylacetic acid ester, swainsonine, lometrexol, dexrazoxane, methioninase,2′-cyano-2′-deoxy-N⁴-palmitoyl-1-B-D-arabino furanosyl cytosine and3-aminopyridine-2-carboxaldehyde thiosemicarbazone.

“HMG-CoA reductase inhibitors” refers to inhibitors of3-hydroxy-3-methylglutaryl-CoA reductase. Examples of HMG-CoA reductaseinhibitors that may be used include but are not limited to lovastatin(MEVACOR®; see U.S. Pat. Nos. 4,231,938, 4,294,926 and 4,319,039),simvastatin (ZOCOR®; see U.S. Pat. Nos. 4,444,784, 4,820,850 and4,916,239), pravastatin (PRAVACHOL®; see U.S. Pat. Nos. 4,346,227,4,537,859, 4,410,629, 5,030,447 and 5,180,589), fluvastatin (LESCOL®;see U.S. Pat. Nos. 5,354,772, 4,911,165, 4,929,437, 5,189,164,5,118,853, 5,290,946 and 5,356,896) and atorvastatin (LIPITOR®; see U.S.Pat. Nos. 5,273,995, 4,681,893, 5,489,691 and 5,342,952). The structuralformulas of these and additional HMG-CoA reductase inhibitors that maybe used in the instant methods are described at page 87 of M. Yalpani,“Cholesterol Lowering Drugs”, Chemistry & Industry, pp. 85-89 (5 Feb.1996) and U.S. Pat. Nos. 4,782,084 and 4,885,314. The term HMG-CoAreductase inhibitor as used herein includes all pharmaceuticallyacceptable lactone and open-acid forms (i.e., where the lactone ring isopened to form the free acid) as well as salt having inhibitoryactivity, and therefore the use of such salts, esters, open-acid andlactone forms in cluster forms of compounds which have HMG-CoA reductaseare within the scope of this invention.

“Prenyl-protein transferase inhibitor” refers to a compound whichinhibits any one or any combination of the prenyl-protein transferaseenzymes, including farnesyl-protein transferase (FPTase),geranylgeranyl-protein transferase type I (GGPTase-1), andgeranylgeranyl-protein transferase type-II (GGPTase-II, also called RabGGPTase).

Examples of prenyl-protein transferase inhibitors can be found in thefollowing publications and patents: WO 96/30343, WO 97/18813, WO97/21701, WO 97/23478, WO 97/38665, WO 98/28980, WO 98/29119, WO95/32987, U.S. Pat. No. 5,420,245, U.S. Pat. No. 5,523,430, U.S. Pat.No. 5,532,359, U.S. Pat. No. 5,510,510, U.S. Pat. No. 5,589,485, U.S.Pat. No. 5,602,098, European Patent Publ. 0 618 221, European PatentPubl. 0 675 112, European Patent Publ. 0 604 181, European Patent Publ.0 696 593, WO 94/19357, WO 95/08542, WO 95/11917, WO 95/12612, WO95/12572, WO 95/10514, U.S. Pat. No. 5,661,152, WO 95/10515, WO95/10516, WO 95/24612, WO 95/34535, WO 95/25086, WO 96/05529, WO96/06138, WO 96/06193, WO 96/16443, WO 96/21701, WO 96/21456, WO96/22278, WO 96/24611, WO 96/24612, WO 96/05168, WO 96/05169, WO96/00736, U.S. Pat. No. 5,571,792, WO 96/17861, WO 96/33159, WO96/34850, WO 96/34851, WO 96/30017, WO 96/30018, WO 96/30362, WO96/30363, WO 96/31111, WO 96/31477, WO 96/31478, WO 96/31501, WO97/00252, WO 97/03047, WO 97/03050, WO 97/04785, WO 97/02920, WO97/17070, WO 97/23478, WO 97/26246, WO 97/30053, WO 97/44350, WO98/02436, and U.S. Pat. No. 5,532,359.

For an example of the role of a prenyl-protein transferase inhibitor onangiogenesis see European J. of Cancer, Vol. 35, No. 9, pp. 1394-1401(1999).

“Agents that interfere with cell cycle checkpoints” refer to compoundsthat inhibit protein kinases that transduce cell cycle checkpointsignals, thereby sensitizing the cancer cell to DNA damaging agents.Such agents include inhibitors of ATR, ATM, the Chk1 and Chk2 kinasesand cdk and cdc kinase inhibitors and are specifically exemplified by7-hydroxystaurosporin, flavopiridol, CYC202 (Cyclacel) and BMS-387032.

“Inhibitors of cell proliferation and survival signaling pathway” referto pharmaceutical agents that inhibit cell surface receptors and signaltransduction cascades downstream of those surface receptors. Such agentsinclude inhibitors of EGFR (for example gefitinib and erlotinib),inhibitors of ERB-2 (for example trastuzumab), inhibitors of IGFR,inhibitors of cytokine receptors, inhibitors of MET, inhibitors of PI3K(for example LY294002), serine/threonine kinases (including but notlimited to inhibitors of Akt such as described in WO 02/083064, WO02/083139, WO 02/083140 and WO 02/083138), inhibitors of Raf kinase (forexample BAY43-9006), inhibitors of MEK (for example CI-1040 andPD-098059) and inhibitors of mTOR (for example Wyeth CCI-779). Suchagents include small molecule inhibitor compounds and antibodyantagonists.

“Apoptosis inducing agents” include activators of TNF receptor familymembers (including the TRAIL receptors).

The invention also encompasses combinations with NSAID's which areselective COX-2 inhibitors. For purposes of this specification NSAID'swhich are selective inhibitors of COX-2 are defined as those whichpossess a specificity for inhibiting COX-2 over COX-1 of at least 100fold as measured by the ratio of IC₅₀ for COX-2 over IC₅₀ for COX-1evaluated by cell or microsomal assays. Such compounds include, but arenot limited to those disclosed in U.S. Pat. No. 5,474,995, U.S. Pat. No.5,861,419, U.S. Pat. No. 6,001,843, U.S. Pat. No. 6,020,343, U.S. Pat.No. 5,409,944, U.S. Pat. No. 5,436,265, U.S. Pat. No. 5,536,752, U.S.Pat. No. 5,550,142, U.S. Pat. No. 5,604,260, U.S. Pat. No. 5,698,584,U.S. Pat. No. 5,710,140, WO 94/15932, U.S. Pat. No. 5,344,991, U.S. Pat.No. 5,134,142, U.S. Pat. No. 5,380,738, U.S. Pat. No. 5,393,790, U.S.Pat. No. 5,466,823, U.S. Pat. No. 5,633,272, and U.S. Pat. No.5,932,598, all of which are hereby incorporated by reference.

Inhibitors of COX-2 that are particularly useful in the instant methodof treatment are: 3-phenyl-4-(4-(methylsulfonyl)phenyl)-2-(5H)-furanone;and5-chloro-3-(4-methylsulfonyl)phenyl-2-(2-methyl-5-pyridinyl)pyridine;

or a pharmaceutically acceptable salt thereof.

Compounds that have been described as specific inhibitors of COX-2 andare therefore useful in the present invention include, but are notlimited to: parecoxib, CELEBREX® and BEXTRA® or a pharmaceuticallyacceptable salt thereof.

As used above, “integrin blockers” refers to compounds which selectivelyantagonize, inhibit or counteract binding of a physiological ligand tothe α_(v)β₃ integrin, to compounds which selectively antagonize, inhibitor counteract binding of a physiological ligand to the α_(v)β₅ integrin,to compounds which antagonize, inhibit or counteract binding of aphysiological ligand to both the α_(v)β₃ integrin and the α_(v)β₅integrin, and to compounds which antagonize, inhibit or counteract theactivity of the particular integrin(s) expressed on capillaryendothelial cells. The term also refers to antagonists of the αvβ₆,αvβ₈, α₁β₁, α₂β₁α₅β₁, α₆β₁ and α₆β₄ integrins. The term also refers toantagonists of any combination of α_(v)β₃, α_(v)β₅, α_(v)β₆, α_(v)β₈,α₁β₁, α₂β₁, α₅β₁, α₆β₁ and α₆β₄ integrins.

Some specific examples of tyrosine kinase inhibitors includeN-(trifluoromethylphenyl)-5-methylisoxazol-4-carboxamide,3-[(2,4-dimethylpyrrol-5-yl)methylidenyl)indolin-2-one,17-(allylamino)-17-demethoxygeldanamycin,4-(3-chloro-4-fluorophenylamino)-7-methoxy-6-[3-(4-morpholinyl)propoxyl]quinazoline,N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)-4-quinazolinamine,BIBX1382,2,3,9,10,11,12-hexahydro-10-(hydroxymethyl)-10-hydroxy-9-methyl-9,12-epoxy-1H-diindolo[1,2,3-fg:3′,2′,1′-kl]pyrrolo[3,4-i][1,6]benzodiazocin-1-one, SH268, genistein,STI571, CEP2563,4-(3-chlorophenylamino)-5,6-dimethyl-7H-pyrrolo[2,3-d]pyrimidinemethanesulfonate, 4-(3-bromo-4-hydroxyphenyl)amino-6,7-dimethoxyquinazoline,4-(4′-hydroxyphenyl)amino-6,7-dimethoxyquinazoline, SU6668, STI571A,N-4-chlorophenyl-4-(4-pyridylmethyl)-1-phthalazinamine, and EMD121974.

Combinations with compounds other than anti-cancer compounds are alsoencompassed in the instant methods. For example, combinations of theinstantly claimed compounds with PPAR-γ (i.e., PPAR-gamma) agonists andPPAR-δ (i.e., PPAR-delta) agonists are useful in the treatment ofcertain malingnancies. PPAR-γ and PPAR-δ are the nuclear peroxisomeproliferator-activated receptors γ and δ. The expression of PPAR-γ onendothelial cells and its involvement in angiogenesis has been reportedin the literature (see J. Cardiovasc. Pharmacol. 1998; 31:909-913; J.Biol. Chem. 1999; 274:9116-9121; Invest. Opthalmol. Vis. Sci. 2000;41:2309-2317). More recently, PPAR-γ agonists have been shown to inhibitthe angiogenic response to VEGF in vitro; both troglitazone androsiglitazone maleate inhibit the development of retinalneovascularization in mice. (Arch. Ophthamol. 2001; 119:709-717).Examples of PPAR-γ agonists and PPAR-γ/α agonists include, but are notlimited to, thiazolidinediones (such as DRF2725, CS-011, troglitazone,rosiglitazone, and pioglitazone), fenofibrate, gemfibrozil, clofibrate,GW2570, SB219994, AR-H039242, JTT-501, MCC-555, GW2331, GW409544,NN2344, KRP297, NP0110, DRF4158, NN622, GI262570, PNU182716, DRF552926,2-[(5,7-dipropyl-3-trifluoromethyl-1,2-benzisoxazol-6-yl)oxy]-2-methylpropionicacid (disclosed in U.S. Ser. No. 09/782,856), and2(R)-7-(3-(2-chloro-4-(4-fluorophenoxy)phenoxy)propoxy)-2-ethylchromane-2-carboxylicacid (disclosed in U.S. Ser. No. 60/235,708 and 60/244,697).

The compounds of the instant invention may also be administered incombination with an inhibitor of inherent multidrug resistance (MDR), inparticular MDR associated with high levels of expression of transporterproteins. Such MDR inhibitors include inhibitors of p-glycoprotein(P-gp), such as LY335979, XR9576, OC144-093, R101922, VX853 and PSC833(valspodar).

A compound of the instant invention may be employed in conjunction withanti-emetic agents to treat nausea or emesis, including acute, delayed,late-phase, and anticipatory emesis, which may result from the use of acompound of the present invention, alone or with radiation therapy. Forthe prevention or treatment of emesis, a compound of the instantinvention may be used in conjunction with other anti-emetic agents,especially neurokinin-1 receptor antagonists, 5HT3 receptor antagonists,such as ondansetron, granisetron, tropisetron, and zatisetron, GABABreceptor agonists, such as baclofen, a corticosteroid such as Decadron(dexamethasone), Kenalog, Aristocort, Nasalide, Preferid, Benecorten orothers such as disclosed in U.S. Pat. Nos. 2,789,118, 2,990,401,3,048,581, 3,126,375, 3,929,768, 3,996,359, 3,928,326 and 3,749,712, anantidopaminergic, such as the phenothiazines (for exampleprochlorperazine, fluphenazine, thioridazine and mesoridazine),metoclopramide or dronabinol. In an embodiment, an anti-emesis agentselected from a neurokinin-1 receptor antagonist, a 5HT3 receptorantagonist and a corticosteroid is administered as an adjuvant for thetreatment or prevention of emesis that may result upon administration ofthe instant compounds.

Neurokinin-1 receptor antagonists of use in conjunction with thecompounds of the instant invention are fully described, for example, inU.S. Pat. Nos. 5,162,339, 5,232,929, 5,242,930, 5,373,003, 5,387,595,5,459,270, 5,494,926, 5,496,833, 5,637,699, 5,719,147; European PatentPublication Nos. EP 0 360 390, 0 394 989, 0 428 434, 0 429 366, 0 430771, 0 436 334, 0 443 132, 0 482 539, 0 498 069, 0 499 313, 0 512 901, 0512 902, 0 514 273, 0 514 274, 0 514 275, 0 514 276, 0 515 681, 0 517589, 0 520 555, 0 522 808, 0 528 495, 0 532 456, 0 533 280, 0 536 817, 0545 478, 0 558 156, 0 577 394, 0 585 913, 0 590 152, 0 599 538, 0 610793, 0 634 402, 0 686 629, 0 693 489, 0 694 535, 0 699 655, 0 699 674, 0707 006, 0 708 101, 0 709 375, 0 709 376, 0 714 891, 0 723 959, 0 733632 and 0 776 893; PCT International Patent Publication Nos. WO90/05525, 90/05729, 91/09844, 91/18899, 92/01688, 92/06079, 92/12151,92/15585, 92/17449, 92/20661, 92/20676, 92/21677, 92/22569, 93/00330,93/00331, 93/01159, 93/01165, 93/01169, 93/01170, 93/06099, 93/09116,93/10073, 93/14084, 93/14113, 93/18023, 93/19064, 93/21155, 93/21181,93/23380, 93/24465, 94/00440, 94/01402, 94/02461, 94/02595, 94/03429,94/03445, 94/04494, 94/04496, 94/05625, 94/07843, 94/08997, 94/10165,94/10167, 94/10168, 94/10170, 94/11368, 94/13639, 94/13663, 94/14767,94/15903, 94/19320, 94/19323, 94/20500, 94/26735, 94/26740, 94/29309,95/02595, 95/04040, 95/04042, 95/06645, 95/07886, 95/07908, 95/08549,95/11880, 95/14017, 95/15311, 95/16679, 95/17382, 95/18124, 95/18129,95/19344, 95/20575, 95/21819, 95/22525, 95/23798, 95/26338, 95/28418,95/30674, 95/30687, 95/33744, 96/05181, 96/05193, 96/05203, 96/06094,96/07649, 96/10562, 96/16939, 96/18643, 96/20197, 96/21661, 96/29304,96/29317, 96/29326, 96/29328, 96/31214, 96/32385, 96/37489, 97/01553,97/01554, 97/03066, 97/08144, 97/14671, 97/17362, 97/18206, 97/19084,97/19942 and 97/21702; and in British Patent Publication Nos. 2 266 529,2 268 931, 2 269 170, 2 269 590, 2 271 774, 2 292 144, 2 293 168, 2 293169, and 2 302 689. The preparation of such compounds is fully describedin the aforementioned patents and publications, which are incorporatedherein by reference.

In an embodiment, the neurokinin-1 receptor antagonist for use inconjunction with the compounds of the instant invention is selectedfrom:2-(R)-(1-(R)-(3,5-bis(trifluoromethyl)phenyl)ethoxy)-3-(S)-(4-fluorophenyl)-4-(3-(5-oxo-1H,4H-1,2,4-triazolo)methyl)morpholine,or a pharmaceutically acceptable salt thereof, which is described inU.S. Pat. No. 5,719,147.

A compound of the instant invention may also be administered with anagent useful in the treatment of anemia. Such an anemia treatment agentis, for example, a continuous eythropoiesis receptor activator (such asepoetin alfa).

A compound of the instant invention may also be administered with anagent useful in the treatment of neutropenia. Such a neutropeniatreatment agent is, for example, a hematopoietic growth factor whichregulates the production and function of neutrophils such as a humangranulocyte colony stimulating factor, (G-CSF). Examples of a G-CSFinclude filgrastim.

A compound of the instant invention may also be administered with animmunologic-enhancing drug, such as levamisole, isoprinosine andZadaxin.

A compound of the instant invention may also be useful for treating orpreventing cancer, including bone cancer, in combination withbisphosphonates (understood to include bisphosphonates, diphosphonates,bisphosphonic acids and diphosphonic acids). Examples of bisphosphonatesinclude but are not limited to: etidronate (Didronel), pamidronate(Aredia), alendronate (Fosamax), risedronate (Actonel), zoledronate(Zometa), ibandronate (Boniva), incadronate or cimadronate, clodronate,EB-1053, minodronate, neridronate, piridronate and tiludronate includingany and all pharmaceutically acceptable salts, derivatives, hydrates andmixtures thereof.

Thus, the scope of the instant invention encompasses the use of theinstantly claimed compounds in combination with a second compoundselected from: HDAC inhibitor (e.g. any one or more of the HDACinhibitors described above), an alkylating agent, an antibiotic agent,an antimetabolic agent, a hormonal agent, a plant-derived agent, abiologic agent, a gene therapy agent, an anti-angiogenic agent, adifferentiation inducing agent, a retinoid receptor modulator, acytotoxic/cytostatic agent, an anti-proliferative agent, an HMG-CoAreductase inhibitor, a prenyl-protein transferase inhibitor, an agentthat interferes with cell cycle checkpoints, an inhibitor of cellproliferation and survival signaling, an apoptosis inducing agent, acell growth arrest inducing agent, a bisphosphonate, or any combinationthereof.

Also included in the scope of the claims is a method of treating cancerthat comprises administering a therapeutically effective amount of acompound of Formula I in combination with radiation therapy and/or incombination with a compound selected from: an alkylating agent, anantibiotic agent, an antimetabolic agent, a hormonal agent, aplant-derived agent, a biologic agent, a gene therapy agent, ananti-angiogenic agent, a differentiation inducing agent, a retinoidreceptor modulator, a cytotoxic/cytostatic agent, an anti-proliferativeagent, an HMG-CoA reductase inhibitor, a prenyl-protein transferaseinhibitor, an agent that interferes with cell cycle checkpoints, aninhibitor of cell proliferation and survival signaling, an apoptosisinducing agent, a cell growth arrest inducing agent, a bisphosphonate,or any combination thereof.

The instant invention also includes a pharmaceutical composition usefulfor treating or preventing cancer that comprises a therapeuticallyeffective amount of a compound of Formula I and a compound selectedfrom: an alkylating agent, an antibiotic agent, an antimetabolic agent,a hormonal agent, a plant-derived agent, a biologic agent, a genetherapy agent, an anti-angiogenic agent, a differentiation inducingagent, a retinoid receptor modulator, a cytotoxic/cytostatic agent, ananti-proliferative agent, an HMG-CoA reductase inhibitor, aprenyl-protein transferase inhibitor, an agent that interferes with cellcycle checkpoints, an inhibitor of cell proliferation and survivalsignaling, an apoptosis inducing agent, a cell growth arrest inducingagent, a bisphosphonate, or any combination thereof.

The dosage regimen utilizing the hydroxamic acid derivatives of thepresent invention can be selected in accordance with a variety offactors including type, species, age, weight, sex and the type of cancerbeing treated; the severity (i.e., stage) of the disease to be treated;the route of administration; the renal and hepatic function of thepatient; and the particular compound or salt thereof employed. Anordinarily skilled physician or veterinarian can readily determine andprescribe the effective amount of the drug required to treat, forexample, to prevent, inhibit (fully or partially) or arrest the progressof the disease.

For oral administration, suitable daily dosages are for example betweenabout 5-4000 mg/m² administered orally once-daily, twice-daily or threetimes-daily, continuous (every day) or intermittently (e.g., 3-5 days aweek). For example, when used to treat the desired disease, the dose ofthe hydroxamic acid can range between about 2 mg to about 2000 mg perday.

The hydroxamic acid derivative is administered once daily (QD), ordivided into multiple daily doses such as twice daily (BID), and threetimes daily (TID). For administration once a day, a suitably preparedmedicament would therefore contain all of the needed daily dose. Foradministration twice a day, a suitably prepared medicament wouldtherefore contain half of the needed daily dose. For administrationthree times a day, a suitably prepared medicament would thereforecontain one third of the needed daily dose.

In addition, the administration can be continuous, i.e., every day, orintermittently. The terms “intermittent” or “intermittently” as usedherein means stopping and starting at either regular or irregularintervals. For example, intermittent administration of an HDAC inhibitormay be administration one to six days per week or it may meanadministration in cycles (e.g., daily administration for two to eightconsecutive weeks, then a rest period with no administration for up toone week) or it may mean administration on alternate days.

Typically, an intravenous formulation may be prepared which contains aconcentration of the hydroxamic acid derivative of between about 1.0mg/mL to about 10 mg/mL. In one example, a sufficient volume ofintravenous formulation can be administered to a patient in a day suchthat the total dose for the day is between about 10 and about 1500mg/m².

Subcutaneous formulations, preferably prepared according to procedureswell known in the art at a pH in the range between about 5 and about 12,also include suitable buffers and isotonicity agents, as describedbelow. They can be formulated to deliver a daily dose of HDAC inhibitorin one or more daily subcutaneous administrations, e.g., one, two orthree times each day.

The term “administration” and variants thereof (e.g., “administering” acompound) in reference to a compound of the invention means introducingthe compound or a prodrug of the compound into the system of the animalin need of treatment. When a compound of the invention or prodrugthereof is provided in combination with one or more other active agents(e.g., a cytotoxic agent, etc.), “administration” and its variants areeach understood to include concurrent and sequential introduction of thecompound or prodrug thereof and other agents.

The compounds can also be administered in intranasal form via topicaluse of suitable intranasal vehicles, or via transdermal routes, usingthose forms of transdermal skin patches well known to those of ordinaryskill in that art. To be administered in the form of a transdermaldelivery system, the dosage administration will, of course, becontinuous rather than intermittent throughout the dosage regime. Itshould be apparent to a person skilled in the art that the various modesof administration described herein merely set forth specific embodimentsand should not be construed as limiting the broad scope of theinvention.

The compounds of this invention may be administered to mammals,preferably humans, either alone or, preferably, in combination withpharmaceutically acceptable carriers, excipients or diluents, in apharmaceutical composition, according to standard pharmaceuticalpractice. The compounds can be administered orally or parenterally,including the intravenous, intramuscular, intraperitoneal, subcutaneous,rectal and topical routes of administration.

In another aspect of the invention, the compounds of this invention maybe administered to mammals, preferably humans, by administering to themammal, either alone or, in combination with pharmaceutically acceptablecarriers, excipients or diluents, in a pharmaceutical composition, themetabolic precursor compound of the compound of the instant invention.Such a metabolic precusor compound is described in U.S. Patent Appl.Nos. 60/388,621, filed on Jun. 14, 2002), 60/403,830, filed on Aug. 15,2002) and 60/426,940, filed on Nov. 15, 2002). Compositions comprisingsuch metabolic precursor compounds; which are also inhibitors of KSP,are also described in those U.S. patent applications.

As used herein, the term “composition” is intended to encompass aproduct comprising the specified ingredients in the specific amounts, aswell as any product which results, directly or indirectly, fromcombination of the specific ingredients in the specified amounts.

The pharmaceutical compositions containing the active ingredient may bein a form suitable for oral use, for example, as tablets, troches,lozenges, aqueous or oily suspensions, dispersible powders or granules,emulsions, hard or soft capsules, or syrups or elixirs. Compositionsintended for oral use may be prepared according to any method known tothe art for the manufacture of pharmaceutical compositions and suchcompositions may contain one or more agents selected from the groupconsisting of sweetening agents, flavoring agents, coloring agents andpreserving agents in order to provide pharmaceutically elegant andpalatable preparations. Tablets contain the active ingredient inadmixture with non-toxic pharmaceutically acceptable excipients whichare suitable for the manufacture of tablets. These excipients may be forexample, inert diluents, such as calcium carbonate, sodium carbonate,lactose, calcium phosphate or sodium phosphate; granulating anddisintegrating agents, for example, microcrystalline cellulose, sodiumcrosscarmellose, corn starch, or alginic acid; binding agents, forexample starch, gelatin, polyvinyl-pyrrolidone or acacia, andlubricating agents, for example, magnesium stearate, stearic acid ortalc. The tablets may be uncoated or they may be coated by knowntechniques to mask the unpleasant taste of the drug or delaydisintegration and absorption in the gastrointestinal tract and therebyprovide a sustained action over a longer period. For example, a watersoluble taste masking material such as hydroxypropyl-methylcellulose orhydroxypropylcellulose, or a time delay material such as ethylcellulose, cellulose acetate buryrate may be employed.

Formulations for oral use may also be presented as hard gelatin capsuleswherein the active ingredient is mixed with an inert solid diluent, forexample, calcium carbonate, calcium phosphate or kaolin, or as softgelatin capsules wherein the active ingredient is mixed with watersoluble carrier such as polyethyleneglycol or an oil medium, for examplepeanut oil, liquid paraffin, or olive oil.

Aqueous suspensions contain the active material in admixture withexcipients suitable for the manufacture of aqueous suspensions. Suchexcipients are suspending agents, for example sodiumcarboxymethylcellulose, methylcellulose, hydroxypropylmethyl-cellulose,sodium alginate, polyvinyl-pyrrolidone, gum tragacanth and gum acacia;dispersing or wetting agents may be a naturally-occurring phosphatide,for example lecithin, or condensation products of an alkylene oxide withfatty acids, for example polyoxyethylene stearate, or condensationproducts of ethylene oxide with long chain aliphatic alcohols, forexample heptadecaethyleneoxycetanol, or condensation products ofethylene oxide with partial esters derived from fatty acids and ahexitol such as polyoxyethylene sorbitol monooleate, or condensationproducts of ethylene oxide with partial esters derived from fatty acidsand hexitol anhydrides, for example polyethylene sorbitan monooleate.The aqueous suspensions may also contain one or more preservatives, forexample ethyl, or n-propyl p-hydroxybenzoate, one or more coloringagents, one or more flavoring agents, and one or more sweetening agents,such as sucrose, saccharin or aspartame.

Oily suspensions may be formulated by suspending the active ingredientin a vegetable oil, for example arachis oil, olive oil, sesame oil orcoconut oil, or in mineral oil such as liquid paraffin. The oilysuspensions may contain a thickening agent, for example beeswax, hardparaffin or cetyl alcohol. Sweetening agents such as those set forthabove, and flavoring agents may be added to provide a palatable oralpreparation. These compositions may be preserved by the addition of ananti-oxidant such as butylated hydroxyanisol or alpha-tocopherol.

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water provide the active ingredient inadmixture with a dispersing or wetting agent, suspending agent and oneor more preservatives. Suitable dispersing or wetting agents andsuspending agents are exemplified by those already mentioned above.Additional excipients, for example sweetening, flavoring and coloringagents, may also be present. These compositions may be preserved by theaddition of an anti-oxidant such as ascorbic acid.

The pharmaceutical compositions of the invention may also be in the formof an oil-in-water emulsion. The oily phase may be a vegetable oil, forexample olive oil or arachis oil, or a mineral oil, for example liquidparaffin or mixtures of these. Suitable emulsifying agents may benaturally occurring phosphatides, for example soy bean lecithin, andesters or partial esters derived from fatty acids and hexitolanhydrides, for example sorbitan monooleate, and condensation productsof the said partial esters with ethylene oxide, for examplepolyoxyethylene sorbitan monooleate. The emulsions may also containsweetening, flavoring agents, preservatives and antioxidants.

Syrups and elixirs may be formulated with sweetening agents, for exampleglycerol, propylene glycol, sorbitol or sucrose. Such formulations mayalso contain a demulcent, a preservative, flavoring and coloring agentsand antioxidant.

The pharmaceutical compositions may be in the form of sterile injectableaqueous solutions. Among the acceptable vehicles and solvents that maybe employed are water, Ringer's solution and isotonic sodium chloridesolution.

The sterile injectable preparation may also be a sterile injectableoil-in-water microemulsion where the active ingredient is dissolved inthe oily phase. For example, the active ingredient may be firstdissolved in a mixture of soybean oil and lecithin. The oil solutionthen introduced into a water and glycerol mixture and processed to forma microemulsion.

The injectable solutions or microemulsions may be introduced into apatient's blood stream by local bolus injection. Alternatively, it maybe advantageous to administer the solution or microemulsion in such away as to maintain a constant circulating concentration of the instantcompound. In order to maintain such a constant concentration, acontinuous intravenous delivery device may be utilized. An example ofsuch a device is the Deltec CADD-PLUS™ model 5400 intravenous pump.

The pharmaceutical compositions may be in the form of a sterileinjectable aqueous or oleagenous suspension for intramuscular andsubcutaneous administration. This suspension may be formulated accordingto the known art using those suitable dispersing or wetting agents andsuspending agents which have been mentioned above. The sterileinjectable preparation may also be a sterile injectable solution orsuspension in a non-toxic parenterally acceptable diluent or solvent,for example as a solution in 1,3-butane diol. In addition, sterile,fixed oils are conventionally employed as a solvent or suspendingmedium. For this purpose any bland fixed oil may be employed includingsynthetic mono- or diglycerides. In addition, fatty acids such as oleicacid find use in the preparation of injectables.

Compounds of Formula I may also be administered in the form ofsuppositories for rectal administration of the drug. These compositionscan be prepared by mixing the drug with a suitable non-irritatingexcipient which is solid at ordinary temperatures but liquid at therectal temperature and will therefore melt in the rectum to release thedrug. Such materials include cocoa butter, glycerinated gelatin,hydrogenated vegetable oils, mixtures of polyethylene glycols of variousmolecular weights and fatty acid esters of polyethylene glycol.

For topical use, creams, ointments, jellies, solutions or suspensions,etc., containing the compound of Formula I are employed. (For purposesof this application, topical application shall include mouth washes andgargles.)

The compounds for the present invention can be administered inintranasal form via topical use of suitable intranasal vehicles anddelivery devices, or via transdermal routes, using those forms oftransdermal skin patches well known to those of ordinary skill in theart. To be administered in the form of a transdermal delivery system,the dosage administration will, of course, be continuous rather thanintermittent throughout the dosage regimen. Compounds of the instantinvention may also be delivered as a suppository employing bases such ascocoa butter, glycerinated gelatin, hydrogenated vegetable oils,mixtures of polyethylene glycols of various molecular weights and fattyacid esters of polyethylene glycol.

The present invention also provides methods of using the hydroxamic acidderivatives of the instant invention for inducing terminaldifferentiation, cell growth arrest and/or apoptosis of neoplastic cellsthereby inhibiting the proliferation of such cells. The methods can bepracticed in vivo or in vitro.

In one embodiment, the present invention provides in vitro methods forselectively inducing terminal differentiation, cell growth arrest and/orapoptosis of neoplastic cells, thereby inhibiting proliferation of suchcells, by contacting the cells with an effective amount of any one ormore of the hydroxamic acid derivatives described herein.

In a particular embodiment, the present invention relates to an in vitromethod of selectively inducing terminal differentiation of neoplasticcells and thereby inhibiting proliferation of such cells. The methodcomprises contacting the cells under suitable conditions with aneffective amount of one or more of the hydroxamic acid compoundsdescribed herein.

In another embodiment, the invention relates to an in vitro method ofselectively inducing cell growth arrest of neoplastic cells and therebyinhibiting proliferation of such cells. The method comprises contactingthe cells under suitable conditions with an effective amount of one ormore of the hydroxamic acid compounds described herein.

In another embodiment, the invention relates to an in vitro method ofselectively inducing apoptosis of neoplastic cells and therebyinhibiting proliferation of such cells. The method comprises contactingthe cells under suitable conditions with an effective amount of one ormore of the hydroxamic acid compounds described herein.

In another embodiment, the invention relates to an in vitro method ofinducing terminal differentiation of tumor cells in a tumor comprisingcontacting the cells with an effective amount of any one or more of thehydroxamic acid compounds described herein.

The methods of the instant invention can be practiced in vitro. It isalso contemplated that the methods of selectively inducing terminaldifferentiation, cell growth arrest and/or apoptosis of neoplasticcells, and of inhibiting HDAC will comprise contacting the cells invivo, i.e., by administering the compounds to a subject harboringneoplastic cells or tumor cells in need of treatment.

Thus, the present invention provides in vivo methods for selectivelyinducing terminal differentiation, cell growth arrest and/or apoptosisof neoplastic cells in a subject, thereby inhibiting proliferation ofsuch cells in the subject, by administering to the subject an effectiveamount of any one or more of the hydroxamic acid derivatives describedherein.

In a particular embodiment, the present invention relates to a method ofselectively inducing terminal differentiation of neoplastic cells andthereby inhibiting proliferation of such cells in a subject. The methodcomprises administering to the subject an effective amount of one ormore of the hydroxamic acid derivatives described herein.

In another embodiment, the invention relates to a method of selectivelyinducing cell growth arrest of neoplastic cells and thereby inhibitingproliferation of such cells in a subject. The method comprisesadministering to the subject an effective amount of one or more of thehydroxamic acid derivatives described herein.

In another embodiment, the invention relates to a method of selectivelyinducing apoptosis of neoplastic cells and thereby inhibitingproliferation of such cells in a subject. The method comprisesadministering to the subject an effective amount of one or more of thehydroxamic acid derivatives described herein.

In another embodiment, the invention relates to a method of treating apatient having a tumor characterized by proliferation of neoplasticcells. The method comprises administering to the patient one or more ofthe hydroxamic acid derivatives described herein. The amount of compoundis effective to selectively induce terminal differentiation, induce cellgrowth arrest and/or induce apoptosis of such neoplastic cells andthereby inhibit their proliferation.

EXAMPLES

Examples provided are intended to assist in a further understanding ofthe invention. Particular materials employed, species and conditions areintended to be illustrative of the invention and not limiting of thereasonable scope thereof.

Example 1 Synthesis

The compounds of the instant invention were prepared by the generalmethods outlined in the synthetic Scheme 1, as exemplified below.

Scheme 1 illustrates the synthesis of the use of the free amine ofaminosuberic acid to generate the carbamate derivatives of the presentinvention.

Procedure for Amide Formation (Similar Method Utilized for OtherAmides):

(7S)-tert-Butoxycarbonylamino-7-phenylcarbamoyl-heptanoic Acid MethylEster

To a stirring solution of (2S)-tert-butoxycarbonylamino-octanedioic acid8-methyl ester (130 mg, 0.429 mmol) in dry CH₂Cl₂ (5 mL) was addedaniline (60 μL, 0.658 mmol), HOBT (61 mg, 0.45 mmol) and EDCI (148 mg,0.772 mmol). The reaction was stirred at RT for 16 h, and then dilutedwith EtOAc (30 mL). The solution was washed with 1M HCl (20 mL), sat.NaHCO₃ (20 mL), H₂O (20 mL), and brine (20 mL). The organic layer wasdried, filtered, and concentrated under reduced pressure to yield athick oil, which was purified by column chromatography (Silica gel;hexanes:EtOAc 3:1) to give the product as a white solid. ¹H NMR (CDCl₃)δ 10.30 (br s, 1H), 7.65 (t, J=7.4 Hz, 211), 7.32 (d, J=7.4 Hz, 2H),7.05 (m, 1H), 5.20 (br, d, 1H), 4.40 (m, 1H), 3.70 (s, 3H), 2.26 (t,J=7.0 Hz, 211), 2.0-1.5 (m, 17H). MS (EI): cal'd (MH⁺) 379, exp (MH⁺)379.

Procedure for Boc Deprotection (Similar Method Used for Other RelatedMaterials):

(7S)-Amino-7-phenylcarbamoyl-heptanoic Acid Methyl Ester

To a solution of(7S)-tert-butoxycarbonylamino-7-phenylcarbamoyl-heptanoic acid methylester (31.0 g, 82.0 mmol) was added methanesulfonic acid (11.7 mL, 0.18mol). After stirring for 1.5 hr, the solvent was removed, and theresidue was diluted with chloroform (600 mL). The pH of the solution wasadjusted to pH 8 by the slow addition of sat. Na₂CO₃. The organic phasewas collected and the aqueous layer was extracted with chloroform (2×50mL). The combined solution was dried over Na₂SO₄. Removal of solventprovided the amine which was used without further purification. ¹H NMR(DMSO-d₆) δ 8.01 (s, br, 1H), 7.65 (t, J=7.4 Hz, 2H), 7.32 (d, J=7.4 Hz,2H), 7.05 (m, 1H), 3.90-3.70 (m, 4H), 2.26 (t, J=7.0 Hz, 2H), 2.15 (m,1H), 2.0-1.5 (m, 7H). MS (EI): cal'd (MH⁺) 279, exp (MH⁺) 279.

General Procedure for Carbamate Formation:

To a solution of (7S)-amino-7-phenylcarbamoyl-heptanoic acid methylester was added the appropriate reagent ROC(O)Cl or the amine wasactivated (e.g. isocyanate or CDI) and quenched with the appropriatealcohol. The solution was stirred at RT overnight. The solvent wasremoved and the intermediate was directly used in the next step.

General Procedure for Hydroxamic Acid Formation:

The residue from the previous reaction was dissolved in DMF. To thesolution was added aqueous 50% hydroxylamine in one portion. The mixturewas stirred at RT for 4 days. The reaction solution was concentratedunder reduced pressure, until incipient precipitation of the product.Water was added slowly to the residue to complete the precipitation. Thesolid was collected and triturated with EtOAc. The solid was collectedand dried under high vacuum pump.

[6-Hydroxycarbamoyl-1-(4-phenyl-thiazol-2-ylcarbamoyl)-hexyl]-carbamicAcid Benzyl Ester

¹H NMR (DMSO-d₆) δ 12.20 (br, s, 1H), 10.30 (br, s, 2H), 8.60 (br, s,1H), 7.80-7.00 (m, 11H), 4.82 (s, 2H), 4.06 (m, 1H), 2.06 (t, J=7.0 Hz,2H), 2.0-1.4 (m, 8H). MS (EI): cal'd (MH⁺) 497, exp (MH⁺) 497.

[6-Hydroxycarbamoyl-1-(4-phenyl-thiazol-2-ylcarbamoyl)-hexyl]-carbamicAcid tert-butyl Ester

¹H NMR (DMSO-d₆) δ 10.30 (br, s, 2H), 7.80-7.00 (m, 6H), 4.06 (m, 1H),2.06 (t, J=7.0 Hz, 2H), 2.0-1.4 (m, 17H). MS (EI): cal'd (MH⁺) 463, exp(MH⁺) 463.

(6-Hydroxycarbamoyl-1-phenylcarbamoyl-hexyl)-carbamic Acid Tert-ButylEster

¹H NMR (DMSO-d₆) δ 10.30 (br, s, 2H), 8.60 (br, s, 2H), 7.40 (d, J=7.4Hz, 2H), 7.20 (t, J=7.4 Hz, 2H), 6.96 (t, J=7.4 Hz, 1H), 4.06 (m, 1H),2.06 (t, J=7.0 Hz, 2H), 2.0-1.4 (m, 171). MS (EI): cal'd (MH⁺) 380, exp(MH⁺) 380.

(6-Hydroxycarbamoyl-1-phenylcarbamoyl-hexyl)-carbamic Acid2-morpholin-4-yl-ethyl Ester

¹H NMR (CD₃OD): δ 7.58 (d, J=7.4 Hz, 2H), 7.31 (t, J=7.4 Hz, 2H), 7.10(t, J=7.4 Hz, 1H), 4.48-4.43 (m, 1H), 4.39-4.36 (m, 1H), 4.22-4.19 (m,1H), 4.04-4.01 (m, 1H), 3.83-3.63 (m, 3H), 3.61-3.58 (2H, m), 3.44-3.42(m, 2H), 3.22-3.08 (m, 2H), 2.09 (t, J=7.0 Hz, 2H), 1.86-1.80 (m, 1H),1.77-1.66 (m, 1H), 1.64-1.58 (m, 2H), 1.54-1.35 (m, 4H). MS (EI): cal'd(MH⁺) 437, exp (MH⁺) 437.

(6-Hydroxycarbamoyl-1-phenylcarbamoyl-hexyl)-carbamic Acid2-pyridin-2-yl-ethyl Ester

MS (EI): cal'd (MH⁺) 429, exp (MH⁺) 429.

(6-Hydroxycarbamoyl-1-phenylcarbamoyl-hexyl)-carbamic Acid3-chloro-benzyl Ester

MS (EI): cal'd (MH⁺) 448, exp (MH⁺) 448.

(6-Hydroxycarbamoyl-1-phenylcarbamoyl-hexyl)-carbamic Acid2-pyrrolidin-1-yl-ethyl Ester

MS (EI): cal'd (MH⁺) 421, exp (MH⁺) 421.

Example 2 HDAC Inhibition by Novel Compounds

HDAC1-Flag Assay:

Novel compounds were tested for their ability to inhibit histonedeacetylase, subtype 1 (HDAC1) using an in vitro deacetylation assay.The enzyme source for this assay was an epitope-tagged human HDAC1complex immuno-purified from stably expressing mammalian cells. Thesubstrate consisted of a commercial product containing an acetylatedlysine side chain (Biomol Research Laboratories, Inc., Plymouth Meeting,Pa.). Upon deacetylation of the substrate by incubation with thepurified HDAC1 complex, a fluorophore is produced that is directlyproportional to the level of deacetylation. Using a substrateconcentration at the Km for the enzyme preparation, the deacetylationassay was performed in the presence of increasing concentrations ofnovel compounds to semi-quantitatively determine the concentration ofcompound required for 50% inhibition (IC50) of the deacetylationreaction.

Results:

The IC₅₀ values for the compounds described in Example 1 were determinedaccording to the method set forth above. All of the compounds were ableto inhibit 50% of the deacetylation reaction at a concentration belowabout 150 mM. Several of the compounds were able to inhibit 50% of thedeacetylation reaction at a concentration below about 50 nM. Several ofthe compounds were able to inhibit 50% of the deacetylation reaction ata concentration below about 20 nM. Several compounds were able toinhibit 50% of the deacetylation reaction at a concentration range ofabout 15 and 20 nM. Several compounds were able to inhibit 50% of thedeacetylation reaction at a concentration range of about 10 and 15 nM.Several compounds were able to inhibit 50% of the deacetylation reactionat a concentration range of about 5 and 10 nM. Several compounds wereable to inhibit 50% of the deacetylation reaction at a concentration ofbelow about 10 nM.

Example 3 HDAC Inhibition in Cell Lines

MTS Assay:

The novel compounds of the instant invention were tested for theirability to inhibit proliferation of the murine erythroleukemia cell lineSC9.

The MTS assay, also referred to as the Cell Titer 96 Aqueous OneSolution Cell Proliferation Assay, is a colorimetric method fordetermining the number of viable cells in proliferation, cytotoxicity orchemosensitivity assays. The MTS reagent contains a novel tetrazoliumcompound[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,inner salt] and electron coupling reagent (phenazine ethosulfate;PES).Murine erythroleukemia cells (SC-9) were incubated with vehicle orincreasing concentrations of compound for 48 hours. Cell proliferationwas quantitated by adding a small amount of the MTS reagent directly toculture wells, incubating for 1-4 hours and then recording theabsorbance at 490 nm with a 96-well plate reader. The quantity offormazan product, as measured by 490 nm absorbance, is directlyproportional to the number of living cells in culture.

Results

The results of the SC9-cell based MTS assay from a select group of novelcompounds show that the compounds are able to inhibit cellularproliferation at a concentration below 5000 nM. Several of the compoundsare able to inhibit cellular proliferation at a concentration below 1000nM. Several of the compounds are able to inhibit cellular proliferationat a concentration range of about 500-1000 nM. Several other compoundsare able to inhibit cellular proliferation at a concentration range ofabout 100-500 nM. Several other compounds are able to inhibit cellularproliferation at a concentration of below 100 nM. Several othercompounds are able to inhibit cellular proliferation at a concentrationrange of about 50-100 nM. Several other compounds are able to inhibitcellular proliferation at a concentration of below 50 nM. Several othercompounds are able to inhibit cellular proliferation at a concentrationof below 25 nM. Several other compounds are able to inhibit cellularproliferation at a concentration of below 10 nM.

While this invention has been particularly shown and described withreferences to embodiments thereof, it will be understood by thoseskilled in the art that various changes in form and details may be madetherein without departing from the meaning of the invention described.Rather, the scope of the invention is defined by the claims that follow.

U.S. Provisional Application No. 60/604,469 is incorporated herein byreference in its entirety.

1. A compound represented by the following structural formula:

wherein R¹ is unsubstituted or substituted and selected from phenyl,thiazolyl, C₂-C₁₀ alkenyl, cycloalkyl, C₁-C₁₀ alkyl-C₂-C₁₀ alkenyl,C₁-C₁₀ alkylcycloalkyl, C₁-C₁₀alkylheterocyclyl and C₁-C₁₀alkylheteroaryl; R² is unsubstituted or substituted and selected fromC₂-C₁₀ alkenyl, cycloalkyl, heterocyclyl, heteroaryl, C₁-C₁₀alkyl-C₂-C₁₀ alkenyl, C₁-C₁₀ alkylcycloalkyl, C₁-C₁₀ alkylheterocyclyland C₁-C₁₀ alkylheteroaryl; R³ and R⁴ are independently hydrogen orC₁-C₁₀ alkyl; and n is 4-7; or a stereoisomer, enantiomer, racemate, orpharmaceutically acceptable salt thereof.
 2. The compound according toclaim 1, wherein R¹ and R² are, independently of each other,unsubstituted or substituted with one, two or three substitutentsselected from R^(sub); wherein R^(sub) is independently selected fromC₁-C₁₀ alkyl, C₁-C₁₀ haloalkyl, C₂-C₁₀ alkenyl, cycloalkyl, aryl,heterocyclyl, heteroaryl, C₁-C₁₀ alkyl-C₂-C₁₀ alkenyl, C₁-C₁₀alkylcycloalkyl, C₁-C₁₀ alkylaryl, C₁-C₁₀ alkylheterocyclyl, C₁-C₁₀alkylheteroaryl, halogen, hydroxy, C₁-C₁₀ alkyloxy, C₁-C₁₀ haloalkyloxy,aryloxy, nitro, oxo, —CN, —C(═O)H, —C(═O)OH, amino, N—C₁-C₁₀ alkylamino,N,N-di C₁-C₁₀ alkylamino, N-arylamino, N,N-diarylamino, N—C₁-C₁₀alkyl-N-arylamino, azido, and C(═O)OR wherein R is aryl or C₁-C₁₀ alkyl;and all other substituents and variables are as defined in claim 1; or astereoisomer, enantiomer, racemate, or pharmaceutically acceptable saltthereof.
 3. The compound according to claim 1, wherein R² isunsubstituted or substituted and selected from cyclopropyl, cyclohexyl,thiazolyl, morpholinyl, and pyrrolidinyl; and all other substituents andvariables are as defined in claim 1; or a stereoisomer, enantiomer,racemate, or pharmaceutically acceptable salt thereof.
 4. The compoundaccording to claim 3, wherein R¹ and R² are, independently of eachother, unsubstituted or substituted with one, two or three substitutentsselected from R^(sub); wherein R^(sub) is independently selected fromC₁-C₄ alkyl, C₁-C₄ haloalkyl, C₁-C₄ haloalkyloxy, C₁-C₄ alkyloxy, aryl,halogen and nitro; and all other substituents and variables are asdefined in claim 3; or a stereoisomer, enantiomer, racemate, orpharmaceutically acceptable salt thereof.
 5. The compound according toclaim 1, wherein R³ and R⁴ are hydrogen.
 6. The compound according toclaim 1, represented by the structure of formula IA:

wherein n is 5 or 6; and all other substituents and variables are asdefined in claim 1; or a stereoisomer, enantiomer, racemate, orpharmaceutically acceptable salt thereof.
 7. The compound according toclaim 1, represented by the structure of formula II:

wherein R² and n are as defined in claim 1; R^(sub) is independentlyselected from C₁-C₄ alkyl, C₁-C₄ haloalkyl, C₁-C₄ haloalkyloxy, C₁-C₄alkyloxy, aryl, halogen and nitro; and m is selected from 0, 1, 2 and 3;and all other substituents and variables are as defined in claim 1; or astereoisomer, enantiomer, racemate, or pharmaceutically acceptable saltthereof.
 8. The compound according to claim 7, represented by thestructure of formula IIA:

wherein n is 5 or 6; and all other substituents and variables are asdefined in claim 7; or a stereoisomer, enantiomer, racemate, orpharmaceutically acceptable salt thereof.
 9. A pharmaceuticalcomposition comprising a pharmaceutically effective amount of thecompound according to claim 1, and a pharmaceutically acceptablecarrier.
 10. A compound which is selected from:(S)-[6-Hydroxycarbamoyl-1-(4-phenyl-thiazol-2-ylcarbamoyl)-hexyl]-carbamicacid benzyl ester;(S)-[6-Hydroxycarbamoyl-1-(4-phenyl-thiazol-2-ylcarbamoyl)-hexyl]-carbamicacid tert-butyl ester; and(S)-(6-Hydroxycarbamoyl-1-phenylcarbamoyl-hexyl)-carbamic acidtert-butyl ester; or a stereoisomer, enantiomer, racemate, orpharmaceutically acceptable salt thereof.
 11. A compound which isselected from: (6-Hydroxycarbamoyl-1-phenylcarbamoyl-hexyl)-carbamicacid 2-morpholin-4-yl-ethyl ester;(6-Hydroxycarbamoyl-1-phenylcarbamoyl-hexyl)-carbamic acid2-pyridin-2-yl-ethyl ester; and(6-Hydroxycarbamoyl-1-phenylcarbamoyl-hexyl)-carbamic acid2-pyrrolidin-1-yl-ethyl ester; or a stereoisomer or pharmaceuticallyacceptable salt thereof.
 12. A pharmaceutical composition comprising apharmaceutically effective amount of the compound according to claim 10,and a pharmaceutically acceptable carrier.
 13. A pharmaceuticalcomposition comprising a pharmaceutically effective amount of thecompound according to claim 11, and a pharmaceutically acceptablecarrier.